CN1643162A - Novel HMGA alleles and use of the same as genetic markers for growth, fatness, meat quality and feed efficiency traits - Google Patents

Abstract

Disclosed herein are genetic markers for animal growth, fatness, meat quality, and feed efficiency, methods for identifying such markers, and methods of screening animals to determine those more likely to produce desired growth, fatness, meat quality, and feed efficiency and preferably selecting those animals for future breeding purposes. The markers are based upon the presence or absence of certain polymorphisms in an HMGA nucleotide sequence.

Description

New HMGA allelotrope and as the application of growth, fat quantity, meat and feed conversion ratio traits genetic markers

Background of invention:

Genetic polymorphism is present between the animal individual, also is present between the kind with required feature that obtains by breeding technique.For example, the known early sexual maturity of Chinese variety, the number height of teeming, and the known growth of u.s. variety is rapid, lean ratio is high.Yet the heredity grade of required proterties is often lower, and selects the traditional breeding way of animal not consider heritable variation and the complicated gene interaction that exists fully according to phenotypic variation.

Several research groups have been arranged with the analyzed and researched DNA of pig of restriction fragment length polymorphism (RFLP).Incorporate the Theor.Appl.Genet. such as Jung of reference at this, 77:271-274 (1989) has reported that they use the heritable variation between two pig varieties of RFLP technology demonstration.Proved the polymorphism of swine leukocyte antigen (SLA) I genoid in these kinds.At this Hoganson etc. that incorporates reference at Abstract for Annual Meeting ofMidwestern Section of the American Society of Animal Science, March26-28 has also reported the polymorphism of Chinese Pigs pig major histocompatibility complex (MHC) gene that rflp analysis proved in 1990.At Theor.Appl.Genet., also reported rflp analysis at this Jung etc. that incorporates reference among the 77:271-274 (1989) to SLA I genoid in some boar.The author proposes may be correlated with between the reproduction of results suggest pig SLA/MHC I genoid and pig and the behavioral trait.They further propose to use SLA I class restriction fragment as genetic marker, help us to improve the growth behavior of pig possibly in the future.

Follow the trail of specific favourable genetic alleles and comprise the method for the dna molecular marker of new and tediously long discriminating main effects gene.。Mark may be with to have a main individual gene of imitating chain, also may with a plurality of gene linkages with additional effect.Dna marker has some advantages: be easy to separating and measuring and clear and definite, and dna marker is codominant, can distinguishes heterozygote and homozygote animal clearly.In case set up a Mk system, select so just to be easy to carry out, because after from the animal young or even embryo, obtaining tissue or blood sample, can carry out the detection of dna marker at any time.

The application of acceptor gene hereditary difference has become a vital signs system of selecting.For example, people's such as Rothschild United States Patent (USP) 5,550,024 and 5,374,526 have disclosed the polymorphism that improves relevant pig female hormone receptor gene with the number of teeming, and it incorporates reference at this; U.S. Patent number 5,935,784 have disclosed the polymorphism mark that improves the pig prolactin antagonist acceptor gene relevant with overall breeding potential with the number of teeming.

The quality of raw pork is subjected to the influence of multiple heredity and non-genetic factor.The latter comprises the farm, transports, butchers and processing conditions.The meat scientist has carried out a large amount of research at these factors, thereby has improved the quality of pork greatly.Part Study is at the genetic background of animal, and some researchs have disclosed the importance of inherited genetic factors.This makes industrially recognizes that animal selection breeding and gene engineering can play important effect aspect the pork quality improving.

The information of dna level can help to determine specific major gene, but it also helps to select with regard to the quantitative character that we select simultaneously.Except phenotypic data,, molecular information selects to reply thereby can improving the tolerance range raising of selection.Many researchers from point of theory to such marker assisted selection system (Marker Assisted Selection) (MAS) size of the additional response the program study.Generally speaking, MAS is more useful for heredity grade hangs down the phenotype that reaches measurement phenotype costliness.Although the proterties as growth, fat quantity, meat and feed conversion ratio is not considered usually in this way, with regard to these proterties, applying marking selects still to have very significantly advantage.For example, Meuwissen and goddard have analyzed the influence of MAS to dissimilar proterties.As having the greatest impact of in butchering the proterties of measuring the back, being subjected to of the proterties of meat and the bonding mark information is extra replys and can reach 64%.For this proterties of measuring on living animal of growth traits, this numeral is little relatively, is 8%.But, in case prove the existence of this association, in phenotype test or before selecting animal (vide infra), can use this label information so, expectation can obtain and be up to 38% reply like this.

Really, the optimal path from heredity improvement economic characters is directly to seek relevant dna marker colony to be selected.Can carry out phenotype in the more faunistic animals of the core of breeding agency continuously measures.Because the overwhelming majority in these proterties can only just can obtain complete assessment after butchering, so data can only be taken from selecteed animal and can not derive from potential breeding animal.

Collect these phenotypic datas so that can detect relevant dna marker, and the mark or the test candidate gene that use the experimental population conclusive evidence to differentiate.Significantly mark or gene can directly apply in the system of selection.An advantage of this molecular information is that we can just obtain it when the breeding animal is also very young, and meaning can be just based on the preselected animal of dna marker before finishing the growth behavior test.This is very favorable for overall test and selective system.

We can see from aforesaid content, need to differentiate genetic marker, and it can be used for improveing by the animal of differentiating and selecting to have the improvement proterties at gene level the useful proterties of economically valuable.

An object of the present invention is to provide based on or the coding HMGA nucleotide sequence in genetic marker, it is a sign as the favourable proterties of economically valuables such as growth, fat quantity, meat and feed conversion ratio.

Another object of the present invention provides the detection method of determining whether this genetic marker exists.

Another purpose provides a kind of method of assessing animal, and this method has improved at the selection of required proterties and the accuracy of breeding method.

Another object of the present invention provides a kind of detection method of pcr amplification in addition, and it can determine fast that this marks whether to exist.

Other purposes of the present invention and an advantage part will be described hereinafter and obtain explanation in the book, and can be partly by obviously drawing in the specification sheets, or recognize by implementing the present invention.Objects and advantages of the present invention can realize by means in the claims and the combination that particularly points out.

Summary of the invention

The present invention relates to the to encode discovery of different genes form of nucleotide sequence of HMGA, it can be used for the heredity of following the trail of the animal strain is differentiated and as the genetic marker relevant with the feed conversion ratio proterties with growth, fat quantity, the meat of animal.These genes are all guarded between species and animal, estimate that the not isoallele that discloses is also economical with other herein or produce the meat animal such as the polymorphism of these genes of ox, sheep, chicken etc. relevant.

In order to reach described purpose and according to purpose of the present invention, as comprise and broadly described herein, the invention provides the discovery of different genotype, it provides genetic typing animal and screening animal to have the method for the animal that shows more disadvantageous growth, fat quantity, meat and feed conversion ratio proterties to determine more likely to possess the animal of favourable growth, fat quantity, meat and feed conversion ratio proterties or select to eliminate.Said herein " favourable growth, fat quantity, meat, feed conversion ratio proterties " one of is meant in a plurality of measurable growths, fat quantity, meat, the feed conversion ratio proterties mean value of comparing a given colony, significant improvement (increase or reduce) is arranged, so this information can be used for breeding to obtain one in the homogeneous colony that optimizes aspect growth, fat quantity, class matter, the feed conversion ratio, this can comprise according to required proterties, improves some proterties or reduces other proterties.Understand the example of some economic characters as desire, can be with reference to following document: Sosnicki, A.A., E.R.Wilson, E.B.Sheiss, A.deVries, 1998 " Is there a cost effective way to produce high quality pork? " ReciprocalMeat Conference Proceedings, Vol.51.

Therefore, the invention provides the screening animal and more may produce favourable growth, fat quantity, meat and feed conversion ratio to differentiate, or unlikely produce these favourable growths, fat quantity, meat and feed conversion ratio animal method, so that optimum growh, fat quantity, meat and feed conversion ratio are optimized breeding and selection technology.

The method that detects these proterties generally comprises following steps: 1) obtain biological sample from animal; And 2) analyze 1) middle genomic dna or the protein that obtains, determine to exist which kind of HMGA allelotrope.Also can use the haplotype data, its a series of polymorphisms with the HMGA gene apply in selection or the authentication schemes to maximize the benefit of these marks.

Because some polymorphisms may be the variation that the HMGA Argine Monohydrochloride is formed, or show and have this variation, so detection method even also can comprise determining that the HMGA Argine Monohydrochloride is formed.The method of carrying out such purifying and analysis typically relates to by comprising the means protein isolate of fluorescent-labeled antibody, separation and purification albumen (promptly utilizing reverse-phase HPLC system), and the aminoacid sequence that uses the evaluation of automatic protein matter sequenator to exist.These detect the method for using is stdn and known in this area, and as (eds.) such as Ausubel, ShortProtocols in Molecular Biology Fourth ed.John Wiley and Sons 1999 discloses.

In the embodiment preferred, analyzed hereditary sample.In brief, from animal, obtain the genetic material sample, and analyze this sample to determine whether to contain and the relevant HMGA nucleotide sequence polymorphism of growth, fat quantity, meat, feed conversion ratio of improvement.

Those skilled in the art know, and the sequence difference between the comparison nucleic acid molecule, have multiple technologies to use.These comprise such as restriction fragment length polymorphism analysis, heteroduple analysis, single-strand conformation polymorphism analysis, denatured gradient electrophoresis and temperature gradient electrophoresis.

In the embodiment preferred, polymorphism is a restriction fragment length polymorphism, and its detection may further comprise the steps: the HMGA gene of identifying animal from isolating genetic material; This gene is exposed to the length different restricted fragment of Restriction Enzyme to produce this gene; The separation limit fragment forms restriction map, as separating by electrophoresis or HPLC; And the restriction fragment collection of illustrative plates that relatively obtains from the known HMGA nucleotide sequence that contains or do not contain described mark.If an animal is positive to labeled test, can consider to include such animal in breeding plan.Test is not positive if animal is to the marker gene type, then it can be chosen as him and use.The application of haplotype data can also be combined with the multiple allelomorphos of the different aspect of screening and growth, fat quantity, meat and feed conversion ratio.

In the most preferred embodiment, separate these genes by using the special zone that the amplification of primer and archaeal dna polymerase contains these genes of polymorphism.Also separate the fragment that obtains with the zone of Restriction Enzyme digest amplification then.Show that the RFLP collection of illustrative plates can be undertaken by fragment is simply dyeed, perhaps primer or ribonucleoside triphosphote by using in the mark amplification procedure.

In another embodiment, the present invention has comprised the method for identifying the genetic marker of relevant growth, fat quantity, meat and feed conversion ratio in specific animal population.Breed same kind or cross-fertilize seed or the jenny of similar genetic strain and buck, and determine growth, fat quantity, meat and the feed conversion ratio of each animal.Differentiate one or whole two the allelic polymorphisms of HMGA of each animal, and it is associated with growth, fat quantity, meat and feed conversion ratio.Preferably, use rflp analysis to determine polymorphism.

In another embodiment, the present invention has comprised the method to a kind of specific economic animal arbitrarily grows, the genetic marker of fat quantity, meat and feed conversion ratio is differentiated.Based on the high conservative of this gene in different animals, infer and only to use the above-mentioned conventional sense method can be in different animals this tag application, with grow based on instruction herein, the selection of fat quantity, meat, feed conversion ratio.Female and the buck that breeds same kind or cross-fertilize seed or have similar genetic strain, and determine growth, fat quantity, meat and the feed conversion ratio of each animal and be associated.For other animal that can obtain its sequence, can use the BLAST contrast to determine whether specific allelotrope is similar to an allelotrope that discloses herein.This similar polymorphism is present in other animal and other genes that are closely related.Term " similar polymorphism " should be by BLAST contrast that determine with above-mentioned arbitrary identical polymorphism.

Following term is used to describe the sequence relation between two or more nucleic acid or the polynucleotide: (a) " reference sequences ", (b) " contrast window ", (c) " sequence identity ", (d) " sequence identity ratio " and (e) " essence consistence ".

(a) " reference sequences " used herein is a sequence of determining that is used for sequence contrast basis, refers to the sequence with reference to HMGA herein.A reference sequences can be part or all of a distinguished sequence, the part of full-length cDNA or gene order for example, or all cDNA or gene order.

(b) " contrast window " used herein refers to a continuous and special fragment of a polynucleotide sequence, wherein this polynucleotide sequence can compare with a reference sequences, and wherein this part polynucleotide sequence in the contrast window is compared with reference sequences (it does not add or disappearance), can comprise interpolation or disappearance (promptly, breach), to contrast this two sequences fully.Generally speaking, contrast window will have 20 successive Nucleotide at least on length, also at random can be 30,40,50,100 even more.Those skilled in the art are appreciated that the high similarity of avoiding containing owing to polynucleotide sequence jagged that cause and reference sequences, typically will introduce a breach penalty value, and deduct from the coupling number.

The control methods of well known contrast sequence.The best correlated contrast of sequence can be adopted local homology's algorithm (Local Homology Algorithm), Smith and Waterman, Adv.Appl.Math.2:482 (1981); Homology contrast algorithm (Homology AlignmentAlgorithm), Needleman and Wunsch, J.Mol.Biol.48:443 (1970); Seek like method (Search for Similarity Method) Pearson and Lipman, Proc.Natl.Acad.Sci.85:2444 (1988); And the computerized applications of these algorithms, comprise but non-being limited to: Intelligenetics Mountain View, the CLUSTAL in the PC/Gene program of California exploitation; Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, GAP, BESTFIT, BLAST, FASTA and TFASTA among the Wisconsin Genetics Software Package of USA exploitation; The CLUSTAL program has detailed description in following document: Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet, et al., NucleicAcids Research 16:10881-90 (1988); Huang, et al., ComputerApplications in the Biosciences 8:155-65 (1992); And Pearson, et al., Methods in Molecular Biology 24:307-331 (1994).The BLAST series of programs that can be used for the search of database similarity comprises: be used for BLASTN that nucleotide query sequence and Nucleotide database sequence are compared; Be used for BLASTX that nucleotide query sequence and Protein Data Bank sequence are compared; Be used for BLASTP that protein search sequence and Protein Data Bank sequence are compared; Be used for TBLASTN that protein search sequence and Nucleotide database sequence are compared; And be used for TBLASTX that nucleotide query sequence and Nucleotide database sequence are compared.Referring to Current Protocols in MolecularBiology, Chapter 19, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995).

Except as otherwise noted, sequence identity provided herein/similarity numerical value refers to a complete set of program of using in BLAST 2.0 versions, the numerical value that obtains under parameter is made as default value.Referring to Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).Carrying out the software that BLAST analyzes can openly obtain, for example NCBI ( Http:// www.hcbi.nlm.nih.gov/).

This algorithm at first will be determined " high sub-sequence to " (high scoring sequencepairs) (HSPs), identify that from search sequence a length is the short word of W, during with word contrast of equal length in the database sequence, its coupling or satisfy certain positive number threshold scores T.T is called as neighborhood word score value threshold value (neighborhood word score threshold) (Altschul et al., the same).These initial neighborhood word samplings (neighborhood wordhits) find to contain the seed of its longer HSPs as initial search.As long as accumulation contrast score value can also increase, the word sampling is just extended to both direction along each sequence.For nucleotide sequence, operation parameter M (the award score value of a pair of coupling residue, always＞0) and N (to the punishment score value of mispairing residue, always＜0) calculate the accumulation score value.For aminoacid sequence, utilize one must calculate the accumulation score value by sub matrix (scoring matrix).The word sampling stops when each direction extends in following situation: accumulation contrast score value is than the little X of its maximum value that obtains; Because the correlated accumulation of one or more negative score value residue, the accumulation score value is equal to or less than 0; Perhaps to the end points of a sequence.The parameter W of BLAST algorithm, T and X have determined correlated sensitivity and speed.The default setting that (nucleotide sequence) BLASTN program is used is 11 as word length (W), and expected value (E) is 10, and the drop value is 100, M=5, and N=-4, and contrast two chains.For aminoacid sequence, the default setting that the BLASTP program is used is 3 as word length (W), and expected value (E) is 10, reaches to such an extent that sub matrix is that BLOSUM62 is (referring to Henikoff﹠amp; Henikoff (1989) Proc.Natl.Acad.Sci.USA 89:10915).

Except that sequence of calculation consistence per-cent, the BLAST algorithm also can carry out statistical study (referring to, Karlin ﹠amp for example to the similarity between two sequences; Altschul, Proc.Natl.Acad.Sci.USA 90:5873-5787 (1993)).A kind of measuring method of the similarity that the BLAST algorithm provides is minimum total possibility (smallest sum probability) (P (N)), the possibility of two Nucleotide of its expression or aminoacid sequence random pair.

BLAST searches hypothetical protein matter can be modeled as stochastic sequence.Yet a lot of real protein comprise nonrandom sequence area, and it may be homopolymer sequence (homopolymeric tracts), short period tumor-necrosis factor glycoproteins or be rich in one or more amino acid whose zone.Can between irrelevant protein, contrast these low-complexity zones, although other zone may be different fully.Can use a plurality of low-complexity filters to reduce these low-complexity contrasts.For example, can be used alone or in combination SEG (Wooten and Federhen, Comput.Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput.Chem., 17:191-201 (1993)) low-complexity filter.

(c) " sequence identity " or " consistence " of two nucleic acid or peptide sequence is meant when carrying out the corresponding contrast of maximum with regard to a special contrast window herein, identical residue in two sequences.When in protein, using sequence identity per-cent, think that inconsistent residue position causes difference by conservative aminoacid replacement usually, wherein amino-acid residue is had the amino-acid residue of similar chemical property (such as charged situation and hydrophobicity) to replace by other, does not therefore change the functional property of molecule.Variant on conservative the replacement when sequence, can raise sequence identity per-cent to proofread and correct the conservative character of this replacement.Because being called as, the discrepant sequence of conservative replacement has " sequence similarity " or " similarity ".Those skilled in the art know the method for carrying out this adjustment.Typically, this need with one conservative replace regard part as rather than mispairing completely come score, thereby improved sequence identity per-cent.Therefore, for example, when a same amino acid is given 1 fen, 0 timesharing is given in a non-conservative replacement, so one conservative replace to give divide between 0 to 1.The conservative score that replaces is by calculating, such as can be according to Meyers and Miller, Computer Applic.Biol.Sci., 4:11-17 (1988) for example, as program PC/GENE (Intelligenetics, Mountain View, California, the algorithm of mentioning in USA).

(d) " sequence identity per-cent " refers to by a contrast window is contrasted the numerical value that two correlated sequences of the best are determined, wherein compare with reference sequences (it does not comprise interpolation or disappearance), the polynucleotide sequence part of this contrast window can comprise interpolation or lack (promptly, breach), contrast this two sequences with the best.Calculate this per-cent by the following method: the number of sites of determining identical nucleic acid base in two sequences or amino-acid residue place is counted to determine match bit, match bit is counted multiply by 100 again to calculate sequence identity per-cent divided by all sites number in the contrast window.

(e) " the essence consistence " of term polynucleotide sequence is meant that polynucleotide comprise such one section sequence, one of contrast program of using above-mentioned canonical parameter setting is determined, it has at least 70% sequence identity, preferably at least 80% than a reference sequences, and more preferably at least 90% and at least 95% sequence identity most preferably.Those skilled in the art are known can to consider that codon annexs under the prerequisite of the location of property, amino acid similarity, reading frame etc., can suitably adjust these values with definite two proteinic consistence that nucleotide sequence is coded.For this purpose, the essence consistence of aminoacid sequence is meant that generally the consistence of sequence is at least 60%, or preferably is at least 70%, 80%, 90%, and most preferably is at least 95%.

These programs and algorithm can be determined the similarity between specific polymorphism and the polymorphism as herein described in a target gene.Estimate that this polymorphism also exists in other animal, and can be according to the prompting of this paper in other animal, just can realize by parameter optimization simply to the utilization of this polymorphism.

In addition, also may set up chainly between the allelotrope of the special allelotrope of the different dna markers dna marker relevant with known and specific gene (the HMGA gene of discussing as this paper), it has shown relevant with specific trait.Therefore, under existing situation, utilize one or whole two HMGA genes, select some allelotrope of HMGA mark of correlation by the special allelotrope of selecting the coloured differently body tag, at least in a short time can select to produce the animal of required growth, fat quantity, meat and feed conversion ratio indirectly, or eliminate the animal that may produce non-required growth, fat quantity, meat and feed conversion ratio.This paper said " genetic marker " should only not comprise by detecting the nucleotide polymorphisms that the little satellite means of polymorphism relevant protein variation, linked marker, use disclose, also even comprise that albumen that certification mark shows changes and use the nucleotide polymorphisms of its means that influences growth of animal, fat quantity, meat and feed conversion ratio announcement.

As used herein, a common specific polymorphism is to name with the title of a specific limited enzyme.This does not also mean that the unique channel in this site of identification just is to use this Restriction Enzyme.Those skilled in the art can obtain many databases and resource and for example identify other Restriction Enzymes that can be used for identifying specific polymorphism Http:// darwin.bio.geneseo.edu, it can provide Restriction Enzyme according to the analysis of sequence and polymorphism to be identified.In fact, this paper also shows has multiple different methods can identify specific polymorphism or allelotrope, and it may even not use Restriction Enzyme, but it detects the multi-form of identical heredity or protein group.

Incorporate into as the annexed drawings set forth of this paper specification sheets integral part one embodiment of the invention, and illustrate principle of the present invention with specification sheets.

Description of drawings

Fig. 1 a and b show BanI (a) and NaeI (b) the PCR-RFLP test result of HMGA I.

Fig. 2 shows the HhaI PCR-RFLP test result of HMGA2.

Fig. 3 is the consensus sequence (SEQ ID NO:19) of pig hmga1.

What underscore and boldface letter indicated is two BanI recognition sites.One of them contains single nucleotide polymorphism at 54.NaeI pleomorphism site (GCYGGC) is represented with underscore.

Y=C or T.

Fig. 4 is the consensus sequence of pig hmga2, length: 1168.Three HhaI recognition sites (GCGC) represent with underscore, two highlights simultaneously wherein, and it contains dna polymorphism.SEQ?ID?NO：20。

Frame 1 is the approximate PCR fragment of Mix1.

Frame 2 is approximate PCR fragments of Mix2.

K=G or T

Y=C or T

Fig. 5 is Berkshire and No. 1 chromosomal recombination frequency figure of Yorkshire Hybrid pig (recomb.Frac., Kosambi cM) shown in the table:

Fig. 6 is Berkshire and No. 7 chromosomal genetic maps of Yorkshire Hybrid pig.Property mean value figure (recomb.frac., Kosambi cM).

Fig. 7 has summed up other primer location and the zone of HMGA1.

Fig. 8 is dna sequence dna (contig (contig) 1) length of pig HMGA1: 2484bp.SEQ ID NOS:21-24

Fig. 9 is dna sequence dna (contig 2) length of pig HMGA1: 1103 bp.NOS:25-29

Detailed Description Of The Invention

To describe embodiment of the present invention in detail below with reference to document, explain principle of the present invention with following embodiment.

The present invention relates to have the relevant genetic marker of proterties of economic worth with animal.The allelotrope of this mark representative and growth, fat quantity, meat and feed conversion ratio proterties significant correlation, therefore whether the screening animal carries out by the existence of differentiating one or two the HMGA nucleotide sequence polymorphism relevant with required growth, fat quantity, meat and feed conversion ratio with the method for the animal of determining more may to produce required growth, fat quantity, meat and feed conversion ratio (one or all these proterties) when the invention provides a kind of breeding.

Therefore, the present invention relates to differentiate certain species (breed), plant system (strain), population (population) or genetic marker of colony (group) animal and the method for identifying these marks, can filter out the animal that possesses required growth, fat quantity, meat and feed conversion ratio thus.

The present invention has identified the neomorph of the HMGA nucleotide sequence relevant with animal improvement proterties.In one embodiment of the invention, illustrated by the new pig HMGA1 allelotrope of BanI or the identification of NaeI restriction site relevant with other as fat, growth, meat and/or feed conversion ratio proterties with lower lipid content.In another embodiment, identified that a new HhaI of the HMGA2 gene relevant with growth traits with fat knows allelotrope.In another embodiment, described mark has been shown and has had additive effect (additive effect) together.

Can use any method that can identify that whether this mark exists, for example comprise that single strand conformation polymorphism (SSCP) analysis, base excision sequence scanning (BESS), rflp analysis, heteroduple analysis, denaturing gradient gel electrophoresis and temperature gradient electrophoresis, allelotrope PCR, ligase chain reaction (LCR) directly check order, micrometering preface, nucleic acid hybridization, microarray assay HMGA gene or other HMGA gene linkage sequence.And scope of the present invention has also comprised under the situation that this polymorphism exists the detection to protein conformation or sequence variation.Polymorphism may not be the reason that causes suddenling change and occur, but it indicates the existence of this variation, and can detect the heredity or the protein basis of phenotypic difference simultaneously.

Following content is that the generality that can be used to detect the technology of polymorphism of the present invention is looked back.

Among the present invention, from animal, obtain the genetic material sample.Sample can come autoblood, tissue, seminal fluid etc.General normal use peripheral blood cells is as the source, and genetic material is DNA.The cell that obtains q.s is to provide the DNA for the capacity of analyzing usefulness.Known or definite easily this amount of those skilled in the art.By technology well known by persons skilled in the art DNA isolation from hemocyte.

The separation of nucleic acid and amplification

Genome DNA sample can separate from any source easily, described source comprises saliva, Stomatocyte, hair root, blood, Cord blood (cord blood), amniotic fluid, tissue juice, peritoneal fluid, Chorionic villi, and have nuclear other suitable cell or tissue sample of complete interkinesis (interphase) or metaphase cell.These cells can be taken from solid tissue, as organ or tissue's sample or biopsy fresh or that preserve.Described sample can contain under state of nature not and blended compounds such as biological substance such as sanitas, antithrombotics, damping fluid, fixing agent, nutritive substance, microbiotic.

The method of isolation of genomic DNA is at for example Kirby from multiple these sources, DNAFingerprinting, An Introduction, W.H.Freeman ﹠amp; Describe among the Co.New York (1992).Genomic dna can also separate oneself former generation or secondary cell culture or the transformation cell lines from the cultivation of arbitrary above-mentioned tissue sample.

Can also use the RNA sample of animal.RNA can separate from the tissue of expressing the HMGA gene as described above according to Sambrook et al..RNA can be total cell RNA, mRNA, poly A+RNA or its arbitrary combination.For obtaining best effect, this RNA of purifying, but also can use not purified endochylema RNA.The RNA reverse transcription can be gone out DNA, gained DNA is as amplification template, so that the specific rna transcription product of the indirect amplification of PCR.See that for example Sambrook is the same, Kawasaki et al., Chapter 8 in PCRTechnology, (1992) the same and Berg et al., Hum.Genet.85:655-658 (1990).

Pcr amplification

The most frequently used amplification means is polymerase chain reaction (PCR), as United States Patent(USP) Nos. 4,683, and 195,4,683,202,4,965,188, all incorporate reference at this.If with increase target region in the hemocyte of PCR, should than keeping apart preservation, also should put on clean gloves during processing with being added with in the valve tube of whole blood suction sealing of heparin with other samples.Be to obtain best effect, immediately blood is handled after being preferably in blood sampling; If do not accomplish this point, blood should be stored in 4 ℃ the sealed vessel to using always so.Also can detect the cell in other physiological fluids.When using these body fluid, the cell in the body fluid should be separated with other body fluid components by centrifugal.

Tissue should be earlier with one a disinfectant disposable scalpel and a disinfectant dissecting needle (perhaps using two scalper) tentatively shred in the culture dish of 5mm.The method of removing paraffin from tissue block all has description in multiple professional handbook well-known to those skilled in the art.

For passing through the target nucleic acid sequence in the pcr amplification sample, this sequence must touch the composition in the amplification system.A kind of method of separating target DNA is the thick extracting method that is used for than large sample.In brief, the monocyte in the blood samples, the amnion cell in the amniocyte (amniocyte), the Chorionic villi cell cultivated etc. separate at aseptic Ficoll-Hypaque gradient higher slice (layering) by standard method.Regather the interval cell, and before extracting DNA, in the sterile phosphate buffer salt solution, wash 3 times.If the DNA of test peripheral blood lymphocyte, osmotic shock (will handle precipitation 10 seconds) is carried out in suggestion again in distilled water, if be also shown in the residual red corpuscle that has then, and washed twice again after washing for the first time.The restraining effect that can avoid the heme group in the oxyphorase that PCR is reacted like this.If the PCR test is not carried out immediately, then cell is divided into 10 after sampling ⁶Individual cell portion is deposited in the Eppendorf pipe and-20 ℃ of lyophilizes, is saved to use.

Cell is resuspended in the 50mM Tris-HCl (pH8.3) that has replenished 100 μ g/ml Proteinase Ks, 50mM KCl, 1.5mM MgCl ₂, 0.5%Tween 20, and (per 100 μ l have 10 in the damping fluid of 0.5%NP40 ⁶Individual karyocyte).56 ℃ hatch two hours after, with cell 95 ℃ the heating 10 minutes with inactivated proteases K, move to rapidly then on ice the cooling (freezing suddenly).If there is bigger aggregation, then also need in same damping fluid, to carry out again another time digestion circulation.Take out 10 μ l extracts as amplification.

When extracting DNA from the cell of organizing Chorionic villi cell for example or confluent culture, so according to the varying in size of tissue sample, the above-mentioned amount of buffer that contains Proteinase K is difference to some extent also.Extract was hatched 4-10 hour at 50-60 ℃, handled 10 minutes inactivated proteases at 95 ℃ afterwards.The longer time hatch in the process, hatch 4 hours with original concentration after, need add fresh Proteinase K again.

Sample contains cell content after a little while, and extraction can be finished with reference to following method, Higuchi, " Simple and Rapid Preparation of Samples for PCR ", PCR Technology, Ehrlich, H.A. (ed.), Stockton Press, New York, it incorporates reference into.Can use pcr amplification from the target region in the seldom amount cell (1000-5000) of the single colony of marrow and peripheral blood cells culture.With the cell suspension in the sample at 20 μ l PCR lysis buffer (10mM Tris-HCl (Ph8.3), 50mM KCl, 2.5mM MgCl ₂, the 0.1mg/ml gelatin, 0.45%NP40,0.45%Tween 20) in and frozen until use.In the time that PCR will be carried out, add 0.6 μ l Proteinase K (2mg/ml) to the cell in the PCR lysis buffer.Then sample is heated to about 60 ℃ and hatched 1 hour.Then sample is heated to 95 ℃ of 10 minutes inactivated proteases K and stops digestion, again in cooled on ice.

Also having a kind of relatively convenient extraction to be used for the method for the DNA of PCR below, is according to Miller et al., the salting-out process of the method improvement of Nucleic Acids Res.16:1215 (1988), and described document is incorporated reference into.Separating monocytic cell on the Ficoll-Hypaque gradient.Cell is resuspended in 3ml lysis buffer (10mM Tris-HCl, 400mM Nacl, 2mM Na ₂EDTA, pH8.2) in.The solution that adds the SDS of the Proteinase K of 50 μ l 20mg/ml and 150 μ l 20% then in cell is then 37 ℃ of overnight incubation.Hatch and shake the digestion that test tube promotes sample in the process.If the digestion of Proteinase K is still not exclusively after the overnight incubation (promptly also having the visible fragment), can in system, add the Proteinase K solution of 50 μ l 20mg/ml again, shake gently or overnight incubation on shaking table at 37 ℃ again.After fully digesting, in sample, add NaCl solution and the violent mixing of 1ml 6M.Gained solution centrifugal 15 minutes at 3000rpm.Precipitation contains sedimentary cell protein, and contains DNA in the supernatant liquor.Supernatant liquor is transferred in the 15ml test tube that the 4ml Virahol is housed.Content in the mixing test tube mixes and forms the DNA precipitation of white mutually with alcohol to water gently.The DNA precipitation is shifted out and splashes into to 70% ethanol, mix gently.Again DNA precipitation is taken out from ethanol and at air drying.Adding distilled water in precipitation dissolves it.

The test kit that is used for the extraction high-molecular-weight DNA of PCR comprises Genomic IsolationKit A.S.A.P (Boehringer Mannheim, Indianapolis, Ind.), Genomic DNAIsolation System (GIBCO BRL, Gaithersburg Md.), Elu-Quik DNAPurification Kit (Schleicher ﹠amp; Schuell, Keene, N.H.), DNA extraction Kit (Stratagene, LaJolla, Calif.), TurboGen Isolation Kit (Invitrogen, SanDiego, Calif.) or the like.Generally speaking can before putting into practice method of the present invention, use these test kits to come purify DNA according to manufacturer's explanation.

The concentration of the DNA that extracts and purity can be by determining in the photoabsorption spectrophotometric analysis of 260nm and 280nm duplicate samples such as dilutions.After extracting DNA, can carry out pcr amplification.The PCR round-robin the first step each time need separate the nucleic acid double chain of primer extension acquisition.In case double-stranded separately, next step of PCR is to make chain separately and primer hybridization at the target sequence flank.Next primer can extend to form the complementary copy of target chain.For successfully carrying out pcr amplification, the design primer in case to make a primer with fundamental chain hybridization after, the subchain of extending generation can be with another primer hybridization and as the template of this primer extension after separating with the fundamental chain sex change.The circulation of sex change, hybridization and extension repeats required number of times to obtain the amplification of nucleic acid of aequum.

In the useful especially embodiment of pcr amplification, by reaction system being heated to sufficiently high temperature and carrying out the sufficiently long time to cause double-stranded sex change but do not cause that polysaccharase generation irreversible denaturation divides open chain (to see U.S.Pat.No.4,965,188, incorporate reference into).Typically the temperature that relates to of thermally denature is between 80 ℃ to 105 ℃, and length several seconds time was to several minutes.Yet separately then can comprise in any suitable method physics, chemistry or the zymetology means of chain realize.For example, can or there be the enzyme induction chain of helicase activity to separate with helicase.For example, enzyme RecA has helicase activity under the situation that has ATP to exist.The suitable reaction condition that chain by helicase separates be known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; And Radding, 1982, Ann.Rev.Genetics 16:405-436 all incorporates reference into).

The primer extension that template relies among the PCR is undertaken by polymerizing agent (polymerizing agent) catalysis in the reaction medium that is including suitable salt, metallic cation and pH buffering system under the situation that has 4 kinds of deoxidation ribonucleoside triphosphotes of capacity (generally being dATP, dGTP, dCTP and dTTP).Suitable polymerizing agent is the DNA synthetic enzyme that known catalytic templating relies on.Under some situation, at least a portion of target region possibility Codocyte expressed proteins.Like this, mRNA also can be used for the amplified target zone.Perhaps, PCR also can be used for making up a cDNA library further to increase from RNA, and the initial template of primer extension is RNA.Being suitable for from the polymerizing agent of synthetic complementary copy DNA (cDNA) sequence of RNA template is reversed transcriptive enzyme (RT), as (Tth) archaeal dna polymerase of avian myeloblastosis virus RT, Moloney murine leukemia virus RT, thermus thermophilus (Thermus Thermophilus), a kind of by PerkinElmer Cetus, Inc. exploitation have reverse transcriptase activity and a heat-stable DNA polymerase.Typically, the first time after the initial reverse transcription step, the geneome RNA template was only stayed dna profiling by thermal destruction in the denaturing step process.Be applicable to that the polysaccharase of making template with DNA comprises, for example, intestinal bacteria (E.coli) dna polymerase i or its Klenow fragment, T4 archaeal dna polymerase, Tth polysaccharase and Taq polysaccharase, separation is from a kind of heat-stable DNA polymerase of thermus aquaticus (Thermus aquaticus), from Perkin ElmerCetus, Inc. can buy.A kind of enzyme in back is widely used in the amplification and the order-checking of nucleic acid.The reaction conditions of use known in the art Taq polysaccharase and at Gelfand, 1989, PCR Technology also has description in the same.

Allele specific pcr

Allele specific oligonucleotide PCR can distinguish and have the target region that lacks variation or polymorphism.Select certain allelic pcr amplification primer in conjunction with target sequence.Gibbs, NucleicAcid Res.17:12427-2448 has described this method in (1989).

Allele specific oligonucleotide screening method

Further the diagnosis screening method has used allele specific oligonucleotide (ASO) screening method (allele-specific oligonucleotide screening method), as Saiki etc. described in the Nature 324:163-166 (1986).Any specific allelotrope is produced the oligonucleotide of one or more base-pair mismatch.The ASO screening method detects the mispairing between variant target gene group or pcr amplified dna and the not mutated oligonucleotide, illustrates compared to sudden change oligonucleotide, the oligonucleotide combination of minimizing.Oligonucleotide probe can design come under low stringency condition with allelic two kinds of polymorphism forms can both in conjunction with, and under high stringent condition, in conjunction with its pairing allelotrope.In addition, can control stringent condition, obtain basic binary and reply, promptly will hybridize with that allelotrope, and not hybridize with wild-type allele corresponding to the ASO of a target gene variant form.

The allelotrope detection method of ligase enzyme mediation

Allelotrope by ligase enzyme mediation detects, can contrast test object DNA target region and the family member that do not influence and influence in target region.See Landegren et al., Science 241:107-1080 (1988).Ligase enzyme also is used in Wu et al., check point sudden change in the ligation amplification reaction of describing among the Genomics4:560-569 (1989).Ligation amplification reaction (LAR) has been realized the amplification of specific dna sequence by the connection of many wheel templates dependence continuously, and as Wu, the same and Barany is described in the Proc.Nat.Acad.Sci.88:189-193 (1990).

Denaturing gradient gel electrophoresis

The amplified production that uses the polymerase chain reaction to obtain can be analyzed with denaturing gradient gel electrophoresis.Different allelotrope can be differentiated with the different of DNA electrophoretic migration in solution by relying on the character of unwinding based on sequence.When temperature rising or the increase of sex change intensity, the dna molecular section unwinds, and these sections are called the structural domain that unwinds (melting domain).Each structural domain that unwinds is worked in coordination with down in melting temperature(Tm) different, that base is special (TM) and is unwind.The structural domain length of unwinding is at least 20 base pairs, also can be up to hundreds of base pairs.

Can be with polyacrylamide gel electrophoresis assessment based on the sequence specific different allelic differentiation of structural domain of unwinding, as Chapter 7, Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W.H.Freeman andCo., New York (1992) is described, and its content is incorporated reference into.

Generally speaking, the target region of analyzing by denaturing gradient gel electrophoresis uses the PCR primer amplification of target region flank.Amplification PCR products is with the polyacrylamide gel analysis of linear denatured gradient, as Myers et al., Meth.Enzymol.155:501-527 (1986) and Myers etal., Genomic Analysis, A Practical Approach, K.Davies Ed.IRL PressLimited, Oxford, pp.95-139 (1988) is described, and its content is incorporated reference into.Electrophoresis system maintains and is lower than the unwind temperature of Tm of structural domain of target sequence slightly.

In the method for another kind of denaturing gradient gel electrophoresis, target sequence can be invested one section GC Nucleotide that is called GC folder (GC clamp) earlier, as Chapter 7, Erlich, same as above.Preferably, having 80% Nucleotide in the GC folder at least is guanine or cytosine(Cyt).Preferably, the GC folder reaches 30 bases at least.This method is specially adapted to the target sequence of high Tm.

Usually, by above-mentioned polymerase chain reaction (PCR) amplification target region.An oligonucleotide PCR primer presss from both sides at the GC that is rich in the GC sequence that 5 ' end has 30 bases at least, and it is incorporated into 5 ' end of target region in amplification procedure.The target region of gained amplification carries out electrophoresis under above-mentioned denatured gradient condition.Only differ the position difference that dna fragmentation that base changes moves on gel, as seen by ethidium bromide staining.

Temperature gradient gel elec-trophoresis (TGGE)

Temperature gradient gel elec-trophoresis (TGGE) (TGGE) and denaturing gradient gel electrophoresis are based on identical principle, just denatured gradient is to be produced by temperature contrast, rather than the concentration of chemical denaturant is different and produce.What the TGGE of standard used is the electrophoresis apparatus that has thermograde with electrophoresis path.When sample moves in the gel of chemical modification agent concentration unanimity, its temperature in the face of raising.The method of another kind of TGGE is time-temperature gradient gel electrophoresis (TTGE or tTGGE), and the temperature of the monoblock running gel that promptly raises step by step is to reach same result.Along with sample moves in gel, the temperature of monoblock gel raises, and therefore passes through gel shift with sample, and they run into more and more higher temperature.Sample is prepared, and comprise the pcr amplification that mixes the GC folder, and the demonstration of product is all identical with denaturing gradient gel electrophoresis.

Single-strand conformation polymorphism analysis

The target sequence of HMGA locus or allelotrope can use single-strand conformation polymorphism analysis to distinguish, the difference of base is distinguished in its change according to strand PCR product electrophoretic migration, as Orita et al., Proc.Nat.Acad.Sci.85:2766-2770 (1989) is described.Amplification PCR products can produce as stated above, heating or other method sex change then, thus form single-stranded amplification product.Single-chain nucleic acid is the folding again or formation secondary structure according to base sequence partly.Therefore, the electrophoretic mobility of single-stranded amplification product can detect the difference of base sequence between allelotrope or the target sequence.

The chemistry of base mismatch or Enzymology method cutting

Difference between target sequence can also be by the right difference chemistry cutting area branch of base mismatch, and as Grompe et al., Am.J.Hum.Genet.48:212-222 (1991) is described.In the another kind method, can be by the difference between the right enzyme cutting detection target sequence of base mismatch, as Nelsonet al., Nature Genetics 4:11-18 (1993) is described.In brief, the genetic material from an animal or an influenced family member can be used to produce the free allos hybrid dna two strands of mispairing." allos hybridization " as referred to herein refers to the chain from an animal of comprising of dna double chain, and from the second chain of another one animal, and differentiated animal on the phenotype of interested proterties normally.Do not have mispairing heteroduplex just select can determine may be relevant with the HMGA polymorphism little insertion, disappearance or other polymorphism.

Non-gel systems

Other possible technology comprises non-gel systems, as TaqMan ^TM(Perkin Elmer).In this system, the oligonucleotide PCR primer of the sudden change flank that design will be studied, and should the zone by pcr amplification.Also to design in addition the 3rd oligonucleotide probe with the area hybridization that in the not isoallele of gene, contains the base object variation.5 ' and 3 ' end of this probe is all used fluorochrome label.When selecting these dyestuffs to close on mutually with convenient its, the fluorescence of one of them can not be detected by the fluorescent quenching of another kind of dyestuff.With respect to probe, PCR primer 5 ' end from template under the effect of Taq archaeal dna polymerase begins to extend, and the dyestuff that causes investing the annealing probe cuts by 5 ' nuclease of Taq archaeal dna polymerase.Removed the feasible fluorescence that can detect probe 3 ' end dyestuff of quenching effect like this.If the hybridization of probe and template molecule is incomplete, promptly there is the mispairing of certain form, dyestuff just can not be cut so, so just can distinguish different dna sequence dnas.Therefore have only nucleotide sequence and the complementation fully of its bonded template molecule, just can remove cancellation when oligonucleotide probe.In the reaction mixture, may contain the sequence of two kinds of different probes, each designs at the not isoallele that may exist, thereby can detect two allelotrope in primary first-order equation.

Also have a kind of technology to be called Invader Assay, it comprises the isothermal duplication that depends on fluorescence catalysis release.See Third Wave Technology, Www.twt.com.

Do not rely on the DNA diagnosis of PCR

Can based on polymorphism comprise an animal and family member restriction fragment length polymorphism polymorphism and identify and the chain dna sequence dna of HMGA sequence without amplification step.Hybridization probe generally is by the oligonucleotide of complementary base pairing in conjunction with total length or part target nucleic acid.The strict degree that probe typically depends on hybridization conditions combines with lacking with the complete complementary target sequence of probe sequence.The preferred directly or indirectly mark of probe, the existence that therefore can whether detect target sequence by the existence of detection probes whether.Directly marking method comprises labelled with radioisotope, as with 32P or 35S.The indirect labelling method comprise fluorescence labels, can be in conjunction with the vitamin H complex body of avidin or streptavidin, or peptide or protein tag etc.The visible detection way comprises photoluminescence agent (photoluminescent), texas Red, rhodamine and derivative thereof, red leuco dye, 3,3 ', 5,5 '-tetramethyl benzidine (TMB), fluorescein and derivative thereof, red sulphonyl, Umbelliferone and similar compound or horseradish peroxidase, alkaline phosphatase etc.

Hybridization probe comprise any can with the pig karyomit(e) at HMGA encoding sequence place hybridization nucleotide sequence, therefore determine and one of HMGA gene chain genetic marker, comprise restriction fragment length polymorphism, hypervariable region, repeat element, or the series connection of variable number repeats.Hybridization probe can be any gene or suitable analogue.Other suitable hybridization probe comprises exon fragment or the part of known locations at the cDNA or the gene of karyomit(e) relevant range.

Being preferably used for series connection recross probe of the present invention is under high strict hybridization conditions, discerns the relatively large segmental probe of this locus when a small amount of fragment of identification specific gene seat or reduction stringent condition.

Can use one or more Restriction Enzyme and/or probe and/or primer.The probe of other enzyme, structure and primer can determine that it is also contained in the scope of the present invention by those skilled in the art's normal experiment.

Although methods described herein can be used single Restriction Enzyme and single one group of primer, these methods are not limited thereto.If desired, can use one or more other Restriction Enzyme and/or probe and/or primer.Really in some cases, may preferably use the marker combination that provides special haplotype.In conjunction with this paper provide and quote instruction, can determine other enzyme, the probe and the primer of structure by normal experiment.

According to the present invention, in one or whole two HMGA nucleotide sequences, identified the polymorphism relevant with growth, fat quantity, meat and feed conversion ratio.Whether the existence of these marks, can analyze by the PCRRFLP that uses restriction endonuclease in one embodiment and detect, and because the high homology of polymorphism peripheral region, can use people of the kind, pig or other HMGA sequences Design amplimer, perhaps can use (for example people) design of known HMGA sequence among the GenBank or even the sequences Design that can draw according to interlocking data from contiguous close gene, as instruction and the reference of this paper.Sequence around the polymorphism can help to design other PCR test, and the primer of wherein choosing about 4-30 continuous base from the sequence of adjacent polymorphism is used for the polymerase chain reaction with this zone of increasing greatly before handling with required Restriction Enzyme.Primer needn't be accurately complementary, and it is just passable completely to be equal to sequence substantially.The design of the known pcr amplification primer of those skilled in the art goes through and sees Ausubel (ed.), among " Short Protocols in MolecularBiology, Fourth Edition " John Wiley and Sons 1999 detailed introduction is arranged.It hereinafter is brief description to design of primers.

The design of primers strategy

The widespread use of polymerase chain reaction (PCR) method impels a lot of program aided design of exploitation or selects to be used as the oligonucleotide of PCR primer.Be to pass through free 4 the such programs that obtain in Internet below: the PRIMER (UNIX of the Mark Daly of Whitehead Institute and SteveLincoln exploitation, VMS, DOS and Macintosh), Oligonucleotide Selection Program (the OSP) (UNIX of the Phil Green of WashingtonUniversity in St.Louis and LaDeana Hiller exploitation, VMS, DOS and Macintish), the Amplify (only being used for Macintosh) of the Bill Engels exploitation of the PGEN (only being used for DOS) of Yoshi exploitation and University ofWisconsin.Usually these programs by seeking known heavy complex sequences element bit and optimize T by length and GC content that primer is inferred in analysis _mAssist design of primers.Can also obtain business software, and the primer method is comprised in just promptly in the most common sequence analysis software bag.

Order-checking and PCR primer

Design the suitable sequence that to select a specific recognition target as the oligonucleotide of order-checking or PCR primer, need test this sequence then to get rid of the possibility that this oligonucleotide has stable secondary structure.Oppositely repeating in the sequence can use above-mentioned repeating to differentiate or the folding program of RNA is differentiated (seeing Prediction of Nucleic Acid Structure).If observed possible loop-stem structure, the sequence of primer can move several Nucleotide to minimize possible secondary structure to any direction so.The sequence of oligonucleotide also should compare with the double-stranded sequence of inserting DNA with appropriate carrier.Clearly, sequencing primer and target DNA should have only single coupling.Also the primer that has only a mispairing with non-required target DNA sequence is not selected in suggestion for use.To being used for the PCR primer of amplifying genom DNA, this primer sequence should compare with the sequence in the GenBank database, to have determined whether any significant coupling.If oligonucleotide sequence appears in any known dna sequence dna, perhaps more crucial is in any known repeat element, just should change primer sequence.

Method of the present invention and material also can broader applications with estimate animal DNA, in the genetic typing individual animals and detect hereditary difference in the animal.Especially, the sample of animal gene group DNA can be by contrasting the reference evaluation with one or more, to determine whether to exist the polymorphism of one of HMGA sequence.Preferably, the HMGA sequence of this animal is carried out rflp analysis, and result and contrast are compared.The result of contrast comes from the rflp analysis of another animal one or whole two HMGA sequences, and wherein the polymorphism of the HMGA gene of this animal is known.Similarly, the HMGA genotype of an animal can be determined by the following method: obtain its genome DNA sample, HMGA gene among the DNA is carried out rflp analysis, again with result and contrast contrast.Equally, results of comparison derives from the rflp analysis of the HMGA sequence of another animal.This result carries out genetic typing by determining the polymorphism of HMGA gene to animal.At last, the hereditary difference between the animal can detect by the following method: from least two animals, obtain genome DNA sample, a HMGA nucleotides sequence list differentiate polymorphism existence whether, and comparative result.

These detections are used to differentiate the genetic marker relevant with growth, fat quantity, meat and feed conversion ratio, as mentioned above, and other polymorphisms that may be relevant in the discriminating HMGA gene, and the general scientific analysis of animal gene type and phenotype with further feature.

The example of this paper and method have disclosed some and have been had the gene of polymorphism by discriminating, and described polymorphism is relevant with a kind of favourable proterties, and this proterties can exert an influence to growth, fat quantity, meat and the feed conversion ratio of the animal of carrying this polymorphism.Whether one gene has polymorphism is identified by the single base conversion that causes a restriction site occurring in some allelic form usually.Yet as this paper proof with discussing, certain allelotrope has a plurality of sequence changes, the sign that these changes can detected identical polymorphism (allelotrope).In addition, other genetic marker or gene may be chain with polymorphism as herein described, therefore detect also to relate to gene or the gene fragment of identifying other, but finally still need depend on the genetic analysis of animal to seek identical polymorphism.The present invention expects can contain any detection method based on allelotrope difference sorting disclosed herein and discriminating animal.

In case one skilled in the art will appreciate that and identified a polymorphism and itself and specific proterties set up and get in touch so just have the several different methods can be according to this polymorphism genetic typing (genotype) animal.These alternative testing schemes are well known by persons skilled in the art to Parameter Optimization, and the present invention also expects can contain these contents.

Embodiment

HMGI (the title of HMGA under the rebaptism system; Bustin, 2001) gene family is by two genomic constitutions, and three kinds of its codings are with chromatin Structure and transcribe and control relevant protein (HMG-I ,-Y and C).HMGI/Y albumen is the product of same gene RNA alternative splicing, and HMGIC is then by different genes encodings.

People's HMGIY (HMGA1) assignment of genes gene mapping is (Friedmann et al., 1993) at chromosomal region 6p21 place, and may participate in the adjusting (Reeves and Beckerbauer, 2001) of the genetic expression of cell growth and differentiation.Therefore, HMGI/Y is proteic unusual or cross formation relevant strongly (Hess, 1998 of expressing with multiple cancer; Tallini and DalCin, 1999; Reeves, 2000).Because HMGI/Y albumen plays Transcription in the expression of adipocyte specific gene, during may and breaking up in the adipocyte growth, HMGI/Y play an important role (Melillo et al., 2001).

At karyomit(e) 12q14-12 place, and to illustrate be the site that often causes the chromosome rearrangement of lipoma (lipoma) to people HMGIC (HMGA2) gene in this zone by physical positioning, and described lipoma is mainly formed (Asher et al., 1995) by mature fat cell.These find to have obtained confirmation in the transgenic mice in the HMGIC gene structure territory of expressing brachymemma.Obesity all takes place and unusual lipoma rate occurred frequently (Arlotta et al., 2000) is arranged in these transgenic mices.In contrast, the mouse of HMGIC gene knockout illustrates adult weight and reduces, and mainly influences fatty tissue (Zhou et al.1995).These results show that the variation of HMGA gene may be relevant with the animal tallow amount with the variation of people's obesity.

The HMGA1:PCR-RFLP test

Primer:

Forward (HMGY1)-5 ' AGA AGG AGC CCA GCG AAG T3 ' SEQ ID NO:1

Reverse (HMYS2)-5 ' ACA GTG CTC ACC CAA TGG C3 ' SEQ ID NO:2

The position: two all are arranged in exon

The PCR condition:

Mix1

10XPCR damping fluid 1.0 μ l

MgCl ₂(25mM)???????0.6μl

dNTPs(2.5mM)???????0.5μl

HMGYl(25pmol/μl)??0.1μl

HMYS2(25pmol/μl)??0.1μl

Taq polysaccharase (5U/ μ l) 0.07 μ l

ddH ₂O?????????????7.63μl

Genomic dna 1.0 μ l

In a PCR reaction tubes, Mix1 and DNA are made up.On Mix, cover mineral oil.React by following PCR program: 94 ℃ 3 minutes; Then 94 ℃ 30 seconds, 63.8 ℃ 1 minute, and 72 ℃ 1 minute 30 seconds, such circulation is carried out 36 times; Carry out last 72 ℃ of extensions of 10 minutes then.The PCR reaction product of the taking out 2 μ l desired result with negative control that on 1.6% sepharose, confirms to increase successfully.

Digestion can be carried out according to the following steps:

BanI digestion reaction: NaeI digestion reaction:

PCR product 4.0 μ l PCR products 4.0 μ l

NE damping fluid 4 1.0 μ l NE damping fluids 1 1.0 μ l

BSA(10mg/ml)???0.1μl????BSA(10mg/ml)??0.1μl

BanI(20U/μl)??0.2μl????NaeI(10U/μl)?0.4μl

ddH ₂O?????????4.7μl????ddH ₂O???????4.5μl

Mix PCR product, damping fluid, enzyme and water.Hatched at least 4 hours or spent the night at 37 ℃.Then digestion solution is mixed (2: 5), electrophoresis on 3% NuSieve sepharose with last sample dyestuff (loading dye).

HMGA2:HhaI PCR-RFLP test

Primer:

HMGIC-5：5′ACT?GAA?GAG?ACA?TCC?TCA?CA3′SEQ?ID?NO：3

HMGIC-T1：5′CTA?AAC?CIG?GGA?CIG?TGA?AG3′SEQ?ID?NO：4

The primer of Mix2: 660bp

HMGIC-SF：5′GAT?AGG?ACT?AGA?TAC?AAC?TTA?C3′SEQ?ID?NO：5

HMGIC-T2：5′GGA?TAT?ATT?GCA?TCT?CTG?GC3′SEQ?ID?NO：6

Mix1：250bp

The PCR condition:

Mix1：???????????????????????????Mix2：

10X Promega damping fluid 1.0 μ l 10X Promega damping fluids 1.0 μ l

25mM?MgCl ₂?????????????0.6μl???25mM?MgCl ₂????????????0.6μl

DNTPs mixture (every kind of 2.5mM) 0.5 μ l dNTPs mixture (every kind of 2.5mM) 0.5 μ l

25pmol/μl?HMGIC?5??????0.1μl???25pmol/μl?HMGIC?SF?????0.1μl

25pmol/μl?HMGIC?T1?????0.1μl???25pmol/μl?HMGIC?T2?????0.1μl

The aseptic H of dd ₂The aseptic H of O 7.4 μ l dd ₂O 7.4 μ l

Taq polysaccharase (5U/ μ l) 0.07 μ l Taq polysaccharase (5U/ μ l) 0.07 μ l

Genomic dna (12.5ng/ μ l) 1.0 μ l genomic dnas (12.5ng/ μ l) 1.0 μ l

1, react by following PCR program: 94 ℃ 2 minutes; Then 94 ℃ 30 seconds, 56 ℃ (Mix1) and 52 ℃ (Mix2) 1 minute, again 72 ℃ 1 minute 30 seconds, circulate 35 times; Extended 10 minutes at 72 ℃ at last then.When HMGIC-5 and HMGIC-T2 primer are used for pcr amplification (annealing temperature is 56 ℃), PCR fragment (1.2kb) contains whole two HhaI pleomorphism sites.

2, on 1% sepharose of standard, detect the PCR reaction product of 3 μ l to confirm the desired result of amplification success and negative control.

3, HhaI digestion reaction: contain at each and to add 5 μ l amplified productions in the pipe of DNA.Hatched at least 4 hours or spent the night at 37 ℃.Then reaction mixture is mixed with last sample dyestuff, with sample on whole volumes on 3% agarose gel.

PCR product 5.0 μ l

10X NE damping fluid 4 1.0 μ l

BSA(10mg/ml)?????0.3μl

HhaI enzyme (20U/ μ l) 0.3 μ l

The aseptic H of dd ₂O 3.6 μ l

The recognition site of BanI is represented with underscore and boldface letter among Fig. 2.One of them contains single nucleotide polymorphism at the 54th.

Fig. 1 illustrates the sequence of HMGA, and it has the NaeI pleomorphism site (GCYGGC) that underscore is represented.

Y=C or T

Three HhaI recognition sites (GCGC) all represent with underscore, wherein contain dna polymorphism with two of grey highlight.

Frame 1 is the roughly PCR fragment of Mix1.

Frame 2 is roughly PCR fragments of Mix2.

K=G or T

Y=C or T

We check order and have analyzed pig HMGA1 and the HMGA2 fragment of using polymerase chain reaction (PCR) amplification.The sequence of pig HMGA2 gene fragment has covered exon 5 to 3 ' UTR, at dna level consistence with corresponding human sequence about 79% is shown.Pig HMGA1 gene fragment order has covered exon 6 and 7, at dna level consistence with corresponding human exon sequence about 93% is shown.

We have also identified some mononucleotides and have replaced (SNPs) in pig HMGA1 and HMGA2 gene.Two SNPs that differentiate in the HMGA1 gene lay respectively in BanI and the NaeI Restriction Enzyme recognition site, and two SNPs in addition that differentiate in the HMGA2 gene are positioned at the HhaI recognition site.Developed the PCR RFLP test of these SNPs and tested from Berkshire * Yorkshire 3 generation family and the DNA sample of the animal of the commercial colony of PIC.Utilize the genotype of the PCR RFLP test of above-mentioned DNA sample to carry out QTL and correlation analysis.For fatty correlated character in Berkshire and the Yorkshire hybridization reference family, two HMGA genes all are positioned at the QTL zone.In the animal of several commercial PIC colony, the existence of the allelotrope 1 NaeI polymorphism of the HMGA1 gene back fat with less significantly is relevant.In addition, in these commercial colonies, the HMGA2 genotype is also relevant with growth traits with fat.The combinatory analysis of two genes in this reference family clearly illustrates that two gene pairs fatty characters have additive effect.

These results show, these polymorphisms are relevant with the multiple proterties with important economic worth of producing pig and meat, and utilize these polymorphisms can accurately select to have the animal of required proterties and phenotype in breeding work.

The correlation analysis of HMGA gene in PIC colony

Fell into a trap all animals on August 1st, 2000 and calculated mean value (s.e.) and Sigma P at meat.

All results are drawn by mixed model, and paternal line as influence at random, and is fixedly butchered the date.

LS mean value significance level: α and δ significance level:

Level:

a-b????p＜.3?????????????ap＜.3

c-d????p＜.1?????????????bp＜.1

e-f????p＜.05??????cp＜.05

g-h????p＜.01??????dp＜.01

i-j????p＜.005?????ep＜.005

k-l????p＜.001?????fp＜.001

m-n????p＜.0005????gp＜.0005

o-p????p＜.0001????hp＜.0001

Estimated value has bias (biased).

Geno p: genotypic p value in proterties=paternal line+butcher date+genotypic model.

The minimizing of the error variance that Expl.% σ e2 is caused by " genotype ".

The adduction and the epistatic effect of the mark of the model of α and δ proterties=paternal line+butcher date+ADD+DOM.

The result provides with character value.

Proterties	Describe
??Dirty?wt	Butcher the dirty weight of back carcass
		??hcw	The weight of the firm poultry trunk that kills
??ccw	Kill the cooled weight of back corpse
		??L_binwt	The band bone weight of waist (waist on one side)
??L_blswt	The weight of boning of waist (waist on one side)
		??loinminl	The minolta L objective colour value (objective color score) of waist
??loinmina	The minolta a objective colour value of waist
		??loinminb	The minolta b objective colour value of waist
??Japcs	Subjective measurement: Japanese color value (1-6)
		Meat between girth of a garment homogeneous phase (marbling)	The subjective value (1-5) of the meat between the girth of a garment homogeneous phase of waist
Hardness (firmness)	The subjective value of waist hardness (1-3)

Waist pH	Waist pH in the time of 24 hours
		????h_binwt	The band bone weight of thigh portion (a personal share portion)
????h_blswt	The weight of boning of the inboard muscle of thigh portion (a personal share portion)
		????hamminl	The minolta L objective colour value of thigh portion
????hammina	The minolta a objective colour value of thigh portion
		????hamminb	The minolta b objective colour value of thigh portion
The pH of thigh portion	The pH of thigh portion in the time of 24 hours
		????dripprct	Driploss per-cent after 48 hours (minimizing of example weight)
????hprofat	The respective thickness of back fat that Henessey detects
		????hpromeat	The loin degree of depth that Henessey detects
????Hprorib	The rib thickness that Henessey detects
		????LMprct	Butcher the lean meat per-cent of back carcass
????aloc_f	Aloca respective thickness of back fat P2 position
		????Endwt	The weight of animal during end of test (EOT)
????Days	Fate during end of test (EOT)
		????LDG，g/d	The weight (from being born) that increase every day in the life cycle to end of test (EOT)
????TDG，g/d	The weight that increase every day during test
		????US_MD	Muscle degree of depth during end of test (EOT)

The analysis of the meat of HMGA1 NaeI and the production traits among the US-Landrace

			No. animal			??????????????????????????LSmeans(s.e.)			geno		????????????α		?????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	2 2	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
??marbling	1.72(0.03)	0.62	170	184	38	1.73(0.04)a	1.75(0.05)c	1.60(0.09)bd	0.20		-0.07(0.05)	a	0.06(0.04)	a
															??firmness	2.75(0.07)	1.13	123	108	14	2.81(0.07)e	2.99(0.07)f	2.90(0.17)	0.06		0.05(0.09)		0.09(0.07)	a
??dripprct	2.80(0.09)	1.44	128	111	13	2.55(0.14)ac	2.82(0.15)bg	1.79(0.38)dh	0.02		-0.38(0.20)	b	0.43(0.16)	d
															??hprofat	12.98(0.11)	2.51	215	219	45	12.77(0.21)e	13.40(0.22)f	13.77(0.41)f	0.014		0.50(0.22)	c	0.09(0.17)
??Hpromast	53.08(0.57)	12.7	220	227	51	53.41(0.64)e	53.46(0.67)e	56.15(1.15)f	0.04		1.37(0.59)	c	-0.88(0.47)	b
															??Hprorib	13.20(0.25)	3.9	124	109	17	13.35(0.39)ic	11.89(0.44)j	11.59(0.96)d	0.008		-0.88(0.50)	b	-0.38(0.40)
??LMprct	46.77(0.08)	1.16	119	101	11	46.99(0.14)ea	46.67(0.16)f	46.52(0.36)b	0.10		-0.23(0.18)	a	-0.06(0.14)
															??Days	159.1(0.70)	12.1	136	128	34	156.4(0.82)ai	157.6(0.87)bg	162.1(1.60)jh	0.005		2.84(0.87)	e	-1.14(0.74)	a
??US_MD	60.75(0.38)	7.71	173	195	48	60.53(0.62)c	60.68(0.61)c	62.87(1.10)d	0.12		1.17(0.60)	b	-0.68(0.50)	a

The analysis of the meat of HMGA1 NaeI and the production traits among the US-Large white

			No. animal			????????????????LSmeans(s.e.)			geno		?????????????α		?????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
Hcw	188.5(0.78)	14.5	93	162	87	185.9(1.83)ce	189.2(1.47)d	190.4(1.75)f	0.09		2.26(1.08)	c	0.71(0.98)
															Ccw	186.3(0.82)	14.5	82	150	78	183.6(1.88)ce	187.0(1.52)d	188.4(1.79)f	0.08		2.39(1.13)	c	0.70(0.99)
Dripprct	2.30(0.11)	1.2	30	62	28	2.67(0.30)e	2.47(0.22)e	1.86(0.28)f	0.07		-0.41(0.18)	c	0.14(0.16)
															Hprofat	14.09(0.15)	2.61	86	150	79	13.66(0.39)ai	14.12(0.32)be	14.95(0.37)jf	0.012		0.65(0.22)	e	-0.12(0.19)
Hprorib	13.46(0.37)	3.97	33	58	23	12.93(0.98)e	13.49(0.76)e	15.81(0.99)f	0.06		1.44(0.61)	c	-0.58(0.54)	a
															LMprct	46.09(0.10)	0.96	28	48	20	46.39(0.27)c	46.25(0.21)c	45.72(0.27)d	0.13		-0.33(0.17)	b	0.13(0.15)
aloc_f	13.48(0.17)	3.2	98	170	90	12.95(0.41)ai	13.42(0.32)be	14.40(0.39)jf	0.009		0.73(0.24)	e	-0.17(0.22)
															Hodwt	109.5(0.36)	6.84	98	170	90	107.7(0.90)ac	109.1(0.72)b	109.5(0.87)d	0.17		0.91(0.52)	b	0.33(0.47)
Days	170.2(0.78)	10.6	43	82	58	166.2(1.89)ae	163.8(1.48)b	161.9(1.65)fa	0.09		-2.17(0.98)	c	-0.20(0.93)
															US_MD	58.71(0.38)	6.92	96	160	82	57.60(0.74)ai	58.66(0.59)be	60.64(0.75)jf	0.009		1.52(0.50)	e	-0.31(0.45)

The analysis of the meat of HMGA1 NaeI and the production traits among US-Large white * Duroc

			No. animal			??????????????????????(s.e.)			geno		?????????????α		??????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
Hprofat	15.88(0.16)	3.46	128	245	109	15.41(0.37)c	15.45(0.29)e	16.31(0.39)df	0.06		0.45(0.23)	b	-0.28(0.19)	a
															mloc_f	13.83(0.15)	3.47	148	274	119	13.35(0.32)ci	13.98(0.26)d	14.68(0.36)jc	0.010		0.67(0.22)	e	-0.02(0.18)

The analysis of the meat of HMGA1 NaeI and the production traits in all strains

			No. animal			????????????LSmeans(s.e.)			geno	Line *geno	??????????????α			????????????????δ
																	Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p	p	Proterties (s.e.)	????p		Proterties (s.e.)	???p
Loinmina	6.85(0.03)	1.59	634	853	702	6.90(0.09)c	7.05(0.08)d	7.08(0.07)d	0.12	0.16	0.09(0.05)	b		0.04(0.04)
																	Loinminb	3.36(0.03)	1.3	627	841	695	3.37(0.06)ae	3.44(0.05)b	3.53(0.05)fa	0.09	0.003	0.08(0.04)	c	b	-0.01(0.03)		c
HampH	5.70(0.00)	0.18	398	529	475	5.71(0.01)a	5.69(0.01)fa	5.70(0.01)b	0.02	0.22	-0.00(0.01)			-0.01(0.01)	c
																	Hprofat	14.68(0.09)	4.17	594	791	680	15.42(0.20)ai	15.66(0.17)be	16.06(0.16)jf	0.02	0.12	0.32(0.11)	e	b	-0.05(0.09)		b
Hpromeat	54.16(0.28)	12.9	612	826	694	54.95(0.47)e	55.32(0.40)c	56.10(0.37)fd	0.10	0.72	0.58(0.27)	c		-0.14(0.23)
																	LMprct	46.17(0.06)	2.01	313	388	353	45.98(0.13)ae	45.86(0.11)be	45.60(0.10)f	0.04	0.47	-0.19(0.07)	c		0.04(0.06)
aloc_f	13.23(0.08)	3.91	664	883	733	13.59(0.20)ci	13.90(0.17)de	14.28?(0.16)jf	0.008	0.45	0.34(0.11)	e	a	-0.02(0.09)
																	US_MD	59.04(0.19)	8.37	536	710	679	57.19(0.45)ai	57.77(0.39)be	58.71(0.35)jf	0.006	0.18	0.76(0.24)	e		-0.12(0.20)		a

The analysis of the meat of HMGA2 and the production traits among the US-Landrace

			No. animal			???????????????????LSmeans(s.e.)			geno		????????????α		?????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
Hprofat	12.96(0.16)	2.56	6	60	186	13.83(0.99)a	12.53(0.37)be	13.35(0.27)f	0.07		-0.24(0.49)		-0.71(0.39)	b
															LMprct	46.78(0.08)	1.17	5	51	135	46.07(0.49)ea	47.16(0.19)fg	46.67(0.15)bh	0.01		0.30(0.24)	a	0.53(0.19)	d
Endwt	111.6(0.43)	6.94	6	61	192	107.5(2.81)c	110.4(0.96)e	112.4(0.68)df	0.04		2.48(1.41)	b	0.28(1.11)
															LDG，g/d	683.0(2.98)	48	6	61	192	638.0(23.1)a	661.8(6.93)c	674.2(5.63)bd	0.09		18.08(11.5)	a	3.78(8.66)
TDG，g/d	896.1(5.58)	73.2	3	43	126	850.1(41.0)a	875.2(12.1)e	900.9(9.76)bf	0.08		25.41(20.4)	a	-0.22(15.4)

The analysis of the meat of HMGA2 and the production traits among the US-Large white

			No. animal			????????????????????LSmeans(s.e.)			geno		????????????α		????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
L_binwt	19.78(0.16)	1.72	12	55	47	20.29(0.45)e	19.97(0.25)e	19.26(0.26)f	0.02		-0.52(0.23)	c	0.13(0.20)
															L_blawt	6.68(0.06)	0.59	12	55	47	6.77(0.15)c	6.69(0.09)e	6.49(0.09)df	0.08		-0.14(0.08)	b	0.04(0.07)
Endwt	109.9(0.50)	6.12	12	68	72	109.1(1.85)	109.4(0.93)e	107.2(0.94)f	0.12		-0.94(0.95)		0.80(0.78)
															LDG，g/d	662.4(3.08)	38	12	68	72	670.2(11.2)a	677.3(5.89)i	658.0(5.21)bj	0.009		-6.12(5.67)	a	8.78(4.63)	b
TDG，g/d	863.5(5.50)	65.8	11	61	71	877.7(19.3)a	876.9(9.85)g	848.3(8.78)bh	0.02		-14.7(9.79)	a	9.25(8.00)	a

The analysis of the meat of HMGA2 and the production traits among the US-Duroc

			No. animal			??????????????????????LSmeans(s.e.)			geno		?????????????α		????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
dirty?wt	230.6(1.65)	19.1	50	48	36	227.6(3.36)e	227.8(2.96)e	236.0(3.24)f	0.07		4.20(2.10)	c	-2.68(2.21)	a
															Hprofat	13.49(0.28)	3.1	46	46	34	14.71(0.67)ic	12.58(0.59)j	13.29(0.64)d	0.01		-0.71(0.39)	b	-0.94(0.40)	c
Endwt	107.0(0.86)	9.97	50	49	35	104.6(1.92)c	104.7(1.67)e	109.1(1.93)df	0.09		2.24(1.17)	b	-1.41(1.20)	a
															LDG，g/d	655.4(4.65)	53.9	50	49	35	648.1(10.8)a	636.5(9.60)be	660.9(10.9)bf	0.09		6.40(5.94)	a	-12.0(5.95)	c
TDG，g/d	847.4(8.81)	100	49	48	33	836.9(19.4)a	816.2(17.0)be	860.8(19.5)bf	0.09		11.95(11.1)	a	-21.8(11.2)	b
															US_MD	56.93(0.60)	6.94	50	49	35	55.01(1.03)ec	57.54(0.91)f	57.44(1.05)d	0.08		1.22(0.65)	b	0.88(0.68)	a

The analysis of the meat of HMGA2 and the production traits among US-Large white * Duroc

			No. animal			???????????????????LSmeans(s.e.)			geno		????????????α		????????????δ
															Proterties	Mean number (s.e.)	σ p	11	12	22	11	12	22	p		Proterties (s.e.)	p	Proterties (s.e.)	p
Hpromast	53.77(0.45)	7.57	55	147	83	54.78(1.06)e	51.81(0.68)fi	55.05(0.88)j	0.002		0.13(0.67)		-2.07(0.58)	f
															Hprorib	15.07(0.26)	4.03	48	125	63	13.89(0.71)ce	15.18(0.48)d	15.76(0.61)f	0.09		0.93(0.43)	c	0.24(0.36)
LMprct	45.49(0.09)-	1.37	48	125	63	45.91(0.22)c	45.50(0.15)d	45.87(0.19)c	0.07		-0.02(0.13)		-0.26(0.11)	c
															Days	151.1(0.65)	8.85	40	100	44	152.0(1.29)a	150.6(0.84)e	153.8(1.24)bf	0.08		0.89(0.85)	a	-1.56(0.78)	c
LDG，g/d	684.3(3.35)	57.2	57	151	84	668.0(7.96)a	678.9(4.85)b	672.8(5.82)	0.41		2.40(4.91)		5.68(4.30)	a
															TDG，g/d	883.5(6.72)	76.1	21	62	45	850.3(13.9)c	877.1(8.45)da	862.5(10.1)b	0.17		6.08(8.55)		13.82(7.41)	b
US_MD	60.25(0.59)	6.12	17	51	40	59.61(1.58)a	60.12(0.94)a	61.88(1.09)b	0.35		1.13(0.96)	a	-0.42(0.77)

In reference family, has the transactional analysis between the HMGA gene of fatty character

A.Berkshire and Yorkshire hybridization family F2 are for animal

1) gene frequency

HMGA1

(BanI) percentage frequency percentage frequency

11??????88????????17.85????88???????17.85

12??????232???????47.06????320??????64.91

22??????173???????35.09????493??????100.00

HMGA2 percentage frequency percentage frequency

11??????64????????12.75????64???????12.75

12??????234???????46.61????298??????59.36

22??????204???????40.64????502??????100.00

HMGA2

Percentage frequency Row Pct Col Pct	?????????????????HMGA1
					????11	????12	????22	Sum
	11	????2 ????0.41 ????3.33 ????2.27	????23 ????4.74 ????38.33 ????10.13	????35 ????7.22 ????58.33 ????20.59					????60 ????12.37
12					????44 ????9.07 ????19.64 ????50.00	????106 ????21.86 ????47.32 ????46.70	????74 ????15.26 ????33.04 ????43.53	????224 ????46.19

22	????42 ????8.66 ????20.90 ????47.73	????98 ????20.21 ????48.76 ????43.17	????61 ????12.58 ????30.35 ????35.88	????201 ????41.44
					Sum	????88 ????18.14	????227 ????46.80	????170 ????35.05	????485 ????100.00

B) on last root bone fat (lrib) proterties influence

All the variation with last root bone proterties is relevant with the HMGA2 genotype for HMGA1.The existence appropriateness of HMGA2 allelotrope 2 has increased last root bone fat fat content (relatively 12 and No. 22 genotype).It is relevant strongly with the increase of lipid content that the existence of HMGA1 allelotrope 1 illustrates.

HMGA2 lrib LSMEAN error

11???????????????????3.18575614???????????0.11603336

12???????????????????3.12045322???????????0.04885318

22???????????????????3.22801544???????????0.04984788

HMGA1 lrib LSMEAN error

11???????????????????3.35804436???????????0.07798118

12???????????????????3.16165424???????????0.04771340

22???????????????????3.07576363???????????0.04890591

Last root bone HMGA1 HMGA2 11 12 22 11 3.38 3.21 2.96 12 3.28 3.14 3.00 22 3.44 3.20 3.21

3) to the influence of waist fat (lum) proterties

HMGA1 illustrates relevant with waist fat (lumbar fat) with the HMGA2 genotype.The combined result of HMGA1 and HMGA2 illustrates the tangible additive effect to waist fat.

HMGA2 lum LSMEAN error

11????????????????3.47003685????????????0.13421404

12????????????????3.53704166????????????0.05650774

22????????????????3.55401930????????????0.05765829

HMGA1 lum LSMEAN error

11????????????????3.77507026????????????0.08990991

12????????????????3.55616811????????????0.05501209

22????????????????3.39947934????????????0.05638702

Lum fat HMGA1 HMGA2 11 12 22 11 3.65 3.72 3.31 12 3.69 3.60 3.39 22 3.91 3.63 3.50

C) to the influence of TL (tlip) proterties

The HMGA2 genotype is mutually relevant with the variation of TL (total lipid)

HMGA2 tlip LSMEAN error

11????????????2.88214497?????????????0.23811466

12????????????3.06472896?????????????0.10025272

22????????????3.23158737?????????????0.10229396

HMGA1 tlip LSMEAN error

11???????????3.33719722??????????0.16344925

12???????????3.03313238??????????0.10000771

22???????????3.10162605??????????0.10250722

??????????HMGA1 HMGA2?????11???????12????????22 11????????2.83?????2.56??????3.04 12????????3.25?????2.91??????3.15 22????????3.46?????3.38??????2.99

5) to the influence of the 10th root bone fat fat (trib) proterties

HMGA1 genotype and the 10th root bone fat fat significant correlation.

HMGA2 trib LSMEAN error

11????????????3.00876115?????????0.13088375

12????????????3.08337129?????????0.05507852

22????????????3.11863733?????????0.05619663

HMGA1 trib LSMEAN error

11????????????3.28558871?????????0.08686549

12????????????3.07574337?????????0.05310117

22????????????2.98812120?????????0.05442963

The 10th root bone HMGA1 HMGA2 11 12 22 11 3.05 3.20 2.95 12 3.26 3.11 2.94 22 3.35 3.23 3.02

1.Berkshire and No. 1 chromosomal genetic map of pig of Yorkshire hybridization family is seen Fig. 4

Property average collection of illustrative plates (recomb.frac., Kosambi cM):

9?SW1515?????????????????????????0.0

0.17?????17.3

2?S0316??????????????????????????17.3

0.03?????3.4

11?SWR2300????????????????????????20.8

0.11?????10.9

10?S0008??????????????????????????31.6

0.16?????16.0

4?SW781??????????????????????????47.7

0.04?????4.3

8?S0312??????????????????????????51.9

0.13?????13.4

7?S0331??????????????????????????65.4

0.03?????3.3

1?MC4R???????????????????????????68.6

0.04?????4.0

0?HMGA2??????????????????????????72.6

0.15?????15.2

6?SW974??????????????????????????87.8

0.17?????17.1

3?SW373??????????????????????????104.9

0.22?????23.0

5?SW1301?????????????????????????128.0

* represent that recomb.frac. remains unchanged in this is analyzed

log10_like＝-1425.68

Property special collection of illustrative plates (recomb.frac., Kosambi cM--is female, and is male):

9?SW1515?????????????????????????0.0?????????????????????????0.0

0.19?????19.7????????????????0.15?????14.9

2?S0316??????????????????????????19.7????????????????????????14.9

0.03?????3.2?????????????????0.02?????2.3

11?SWR2300????????????????????????22.9????????????????????????17.2

0.08?????7.6?????????????????0.16?????16.1

10?S0008??????????????????????????30.5????????????????????????33.3

0.16?????16.6????????????????0.14?????14.8

4?SW781??????????????????????????47.1????????????????????????48.1

0.02?????2.0?????????????????0.07?????7.0

8?S0312??????????????????????????49.1????????????????????????55.1

0.22?????23.4????????????????0.04?????4.1

7?S0331??????????????????????????72.5????????????????????????59.2

0.04?????4.5?????????????????0.02?????2.1

1?MC4R???????????????????????????77.0????????????????????????61.3

0.06?????5.6?????????????????0.02?????2.2

0?HMGA2??????????????????????????82.5????????????????????????63.5

0.20?????21.3????????????????0.09?????9.5

6?SW974??????????????????????????103.8???????????????????????73.0

0.20?????21.6????????????????0.13?????13.3

3?SW373??????????????????????????125.4???????????????????????86.3

0.19?????19.4????????????????0.24?????26.4

5?SW1301?????????????????????????144.8???????????????????????112.7

* represent that recomb.frac. remains unchanged in this is analyzed

log10_like＝-1396.97

2.Berkshire and No. 7 chromosomal genetic maps of pig of Yorkshire hybridization family are seen the average collection of illustrative plates of Fig. 5 (recomb.frac., Kosambi cM):

10???S0025????????????????????0.0

0.27???29.7

9???S0064????????????????????29.7

0.17???18.1

8????TNFB????????????????????47.8

0.04???4.4

0???HMGA1????????????????????52.3

0.12???12.5

7???SWR1928??????????????????64.7

0.10????9.7

2???SW2040???????????????????74.4

0.09????9.0

5???SW252????????????????????83.5

0.06????6.1

1???SW632????????????????????89.6

0.05????5.5

4???SW1083???????????????????95.1

0.20???21.6

3???S0101????????????????????116.7

0.21???22.6

6???SW764????????????????????139.3

^*Expression recomb.frac. remains unchanged in this is analyzed

log10_like＝-1441.45

Property special collection of illustrative plates (recomb.frac., Kosambi cM--is female, and is male):

10??S0025????0.0?????0.0????0.30???35.4???0.23???24.9

9??S0064????35.4????24.9???0.14???14.2???0.21???22.1

8??TNFB?????9.6?????47.0???0.06???5.6????0.04???3.5

0??HWGA1????55.2????50.5???0.17???17.8???0.08???7.9

7??SWR1928??72.9????58.4???0.08???7.8????0.12???11.9

2??SW2040???80.7????70.3???0.12???12.1???0.06???5.9

5??SW252????92.9????76.2???0.09???9.0????0.04???3.8

1??SW632????101.8???80.0???0.05???5.0????0.06???6.3

4??SW1083???106.9???86.3???0.21???21.8???0.20???21.3

3??S0101????128.7???107.6??0.25???27.3???0.17???17.8

6??SW764????????????156.0?????????125.5

log10_like＝-1428.65

Other primers of HMGA1 and new sequence

Primer:

HMA1-F1 (forward) 5 ' AAG CAG CCT CCG GTG AGT C3 ' SEQ ID NO:7

HMA1-R1 (forward) 5 ' CAC TTC GCT GGG CTC CTT CT3 ' SEQ ID NO:8

Arrive between the intron 5 (～1800bp, annealing temperature Ta are 65 ℃) at exon 5

HM766F (forward) 5 ' TCT CTA GTT CCT CAT TCC3 ' SEQ ID NO:9

HM766R (forward) 5 ' CCC AAG ACA GAA TAA AAA G3 ' SEQ ID NO:10

The both is positioned at intron 5 (～800bp, Ta are 51.7 ℃)

HM867F (forward) 5 ' CCT CTT GTC ATT TTA CTG TC3 ' SEQ ID NO:11

HM867R (forward) 5 ' ACC CCA CTT TCC TCA ACT3 ' SEQ ID NO:12

Arrive between the intron 6 (～390bp, Ta are 57.4 ℃) at intron 5

HM978F (forward) 5 ' CTC TGC CTC CAC TCT CTA3 ' SEQ ID NO:13

HM978R (forward) 5 ' TGC CAA AGG TGA CAA GAC3 ' SEQ ID NO:14

All be positioned at intron 5 (～1000bp, Ta are 59.3 ℃)

HMAI2F (forward) 5 ' CCA GGA AGG AAA CCA AGG G3 ' SEQ ID NO:15

HMAI2R (forward) 5 ' TGA CTC AGC AAC CTC CAC G3 ' SEQ ID NO:16

Arrive between the intron 7 (～1200bp, Ta are 60 ℃) at exon 7

HMAI2F (forward) 5 ' CCA GGA AGG AAA CCA AGG G3 ' SEQ ID NO:17

HMAI3R (forward) 5 ' TGA CTC AGC AAC CTC CAC G3 ' SEQ ID NO:18

Arrive between the intron 7 (～800bp, Ta are 56 ℃) at exon 7

The PCR condition:

Mix1：

10X PCR damping fluid 1.0 μ l

MgCl ₂(25mM)???????0.6μl

dNTPs(2.5mM)???????0.5μl

HMGY1(25pmol/μl)??0.1μl

HMYS2(25pmol/μl)??0.1μl

Taq polysaccharase (5U/ μ l) 0.07 μ l

ddH ₂O????????????????7.63μl

Genomic dna 1.0 μ l

Combination Mix1 and DNA in the PCR reaction tubes.Above mixture, cover mineral oil.React by following PCR program: 94 ℃ 3 minutes; 94 ℃ of 36 round-robin 30 seconds, Ta1 minute and 72 ℃ 1 minute and 40 seconds; Then 72 ℃ of last extensions of carrying out 10 minutes.The required desired result of the PCR reaction product that on 1.6% sepharose, detects 2 μ l to confirm to increase successfully with negative control.(every cover primer sets is carried out under suitable annealing temperature in identical program, as above-mentioned)

Attention: other SNPs differentiates by sequential analysis.The SNP position is represented with boldface letter (boldfont).

Contig between each kind (contig) sequence is to the consensus sequence of HMGA1 when

Annotate: other SNPS differentiates by sequential analysis.The order-checking of the pig of 4 different varietiess shown have some polymorphisms in the HMGA1 gene DNA sequence.The SNP position is represented with symbol (* ,+) and arrow.Be description below to SNPs.

Table 1: the description that the position of the new SNPs that the sequential analysis by contig 1 and 2 is differentiated and base change

	The position	????SNP	Be positioned at		The position	????SNP	Be positioned at
								Contig 1	????197	????G/T	Intron 5	Contig 1	????1391	????A/G	Intron 5
	????259	????C/T	Intron 5		????1564	????A/C	Intron 5
									????309	????G/A	Intron 5		????1643	????G/A	Intron 5
	????913	????T/C	Intron 5		????1784 ????(BanI)	????C/T	Exon 6
									????1004	????T/G	Intron 5		????1848	????A/C	Intron 6
	????1028	????T/C	Intron 5		????2306 ????(NaeI)	????C/T	Intron 6
									????1065	????G/A	Intron 5		????2375	????T/C	Intron 6
	????1170	????G/A	Intron 5		????123	????C/A	Intron 7
									????1333	????G/A	Intron 5		????762	????C/T	Intron 7

Annotate: contig 2 also has other potential SNPs that can't confirm by sequential analysis.

Reference

Arlotta，P.，Tai，A.K.，Manfioletti，G.，Clifford，C.，Jay，G.，Ono，S.J.2000.Transgenic?mice?expressing?a?truncated?form?of?the?high?mobilitygroup?I-C?protein?develop?adiposity?and?an?abnormally?high?prevalenceof?lipomas.J.Biol.Chem.275，14394-14400.

Ashar，H.R.，Fejzo，M.S.，Tkachenko，A.，Zhou，X.，Fletcher，J.A.，Weremowicz，S.，Morton，C.C.，Chada，K.1995.Disruption?of?thearchitectural?factor?HMGI-C：DNA-binding?AT?hook?motifs?fused?inlipomas?to?distinct?transcriptional?regulatory?domains.Cell?82：57-65.

Bustin，M.，2001.Revised?nomenclature?for?high?mobility?group(HMG)chromosomal?proteins.Trends?Biochem.Sci.26，152-153.

Friedmann，M.，Holth，L.T.，Zoghhbi，H.Y.，Reeves，R.1993.Organization，inducible-expression?and?chromosome?localization?of?thehuman?HMGI(Y)nonhistone?protein?gene.Nucleic?Acids?Res.21，4259-4267.

Hess，J.L.1998.Chromosomal?translocaitons?in?benign?tumors.Am.J.Clin.Path.109，251-261.

Melillo，R.M，，Pierantoni，G.M，，Scala.S.，Battista，S.，Fedele，M.，Stella，A.，De?Biasio，M.C.，Chiappetta，G.，Fidanza，V.，Condorelli，G.，Santoro，M.，Croce，C.M.，Viglietto，G.，Fusco，A.2001.Critical?role?ofthe?HMGI(Y)proteins?in?adipocytic?cell?growth?and?differentiation.Mol.Cell.Biol.21，2485-2495.

Tallini，G.，Dal?Cin，P.1999.HMGI(Y)and?HMGI-C?dysregulation：a?common?occurrence?in?human?tumors.Adv.Anat.Pathod.6，237-246.

Reeves，R.2000.Structure?and?function?of?the?HMGI(Y)family?ofarchitectural?transcription?and?chromatin?structure.Environ.HealthPerspect.108，803-809.

Reeves，R.，Beckerbauer，L.2001.HMGI/Y?proteins：flexibleregulators?of?transcription?and?chromatin?structure.Biochim.Biophy.Acta?1519，13-29.

Zhou，X.，Benson，K.F.，Ashar，H.R.，Chada，K.1995.Mutationresponsible?for?the?mouse?pygmy?phenotype?in?the?developmentallyregulated?factor?HMGI-C.Nature?376，771-774.

Sequence table

＜110〉Iowa State Univ. Research Foundation, Inc.

＜120〉new HMGA allelotrope and as the application of growth, fat quantity, meat and feed conversion ratio traits genetic markers

<130>ISURF?2900

<150>60/364,959

<151>2002-03-15

<160>30

<170>PatentIn?version?3.1

<210>1

<211>19

<212>DNA

<213>porcine

<400>1

agaaggagcc?cagcgaagt????????????????????????????????????????????????19

<210>2

<211>19

<212>DNA

<213>porcine

<400>2

acagtgctca?cccaatggc????????????????????????????????????????????????19

<210>3

<211>20

<212>DNA

<213>porcine

<400>3

actgaagaga?catcctcaca????????????????????????????????????????????20

<210>4

<211>20

<212>DNA

<213>porcine

<400>4

ctaaacctgg?gactgtgaag????????????????????????????????????????????20

<210>5

<211>22

<212>DNA

<213>porcine

<400>5

gataggacta?gatacaactt?ac?????????????????????????????????????????22

<210>6

<211>20

<212>DNA

<213>porcine

<400>6

ggatatattg?catctctggc????????????????????????????????????????????20

<210>7

<211>19

<212>DNA

<213>porcine

<400>7

aagcagcctc?cggtgagtc?????????????????????????????????????????????19

<210>8

<211>20

<212>DNA

<213>porcine

<400>8

cacttcgctg?ggctccttct????????????????????????????????????????????20

<210>9

<211>18

<212>DNA

<213>porcine

<400>9

tctctagttc?ctcattcc??????????????????????????????????????????????18

<210>10

<211>19

<212>DNA

<213>porcine

<400>10

cccaagacag?aataaaaag?????????????????????????????????????????????19

<210>11

<211>20

<212>DNA

<213>porcine

<400>11

cctcttgtca?ttttactgtc????????????????????????????????????????????20

<210>12

<211>18

<212>DNA

<213>porcine

<400>12

accccacttt?cctcaact??????????????????????????????????????????????18

<210>13

<211>18

<212>DNA

<213>porcine

<400>13

ctctgcctcc?actctcta??????????????????????????????????????????????18

<210>14

<211>18

<212>DNA

<213>porcine

<400>14

tgccaaaggt?gacaagac??????????????????????????????????????????????18

<210>15

<211>19

<212>DNA

<213>porcine

<400>15

ccaggaagga?aaccaaggg?????????????????????????????????????????????19

<210>16

<211>19

<212>DNA

<213>porcine

<400>16

tgactcagca?acctccacg?????????????????????????????????????????????19

<210>17

<211>19

<212>DNA

<213>porcine

<400>17

ccaggaagga?aaccaaggg?????????????????????????????????????????????19

<210>18

<211>19

<212>DNA

<213>porcine

<400>18

tgactcagca?acctccacg?????????????????????????????????????????????19

<210>19

<211>703

<212>DNA

<213>porcine

<400>19

ccaacaccta?aaagacctcg?gggccgacca?aaggggagca?aaaacaaggg?cgcygccaag?????60

acccgggtga?ggcttgaagg?ggtggctcct?ggtggaggga?agtgggaagt?aaccccccgc????120

cccctgcaag?cagctgaggg?aggtctggga?aggggtgggc?ttgtcctgat?tctctgcatg????180

ccctttctct?ggtacgtggg?cccgatgggt?cttggctagt?tgaggaaagt?ggggtgatgg????240

ccgaggccta?acttctaggg?ccttgtcttg?cccaggacac?tggggaagtc?aagtcagatg????300

tcccagagct?ttcctggtct?ggagggaggc?cagttgggca?gaatggaggg?ctgttccccc????360

tgggctgaga?tgtcacctcc?cccccaaccc?caggccgcct?gggtcctgag?ggtgggggag????420

caggcaaggc?cagatctaca?gtggcattgg?cctttggaga?agttgttttg?ttttttattt????480

tattttttct?aagacacgac?tcatatcctc?tgagtcacgg?gtgaaggagg?gagtgggggc????540

gtgtgtgtgt?atgttggggt?ggggggcggt?gtggcyggcc?agtcatcccc?agctggactc????600

cggtgggcct?gctgggctga?gagtcccggc?tgcccctccc?tgctcgccct?cgccctccag????660

ggcactggtc?actgcggggc?acccgccatt?gggtgagcac?tgt??????????????????????703

<210>20

<211>1168

<212>DNA

<213>porcine

<400>20

actgaagaga?catcctcaca?gaagtctgca?gaagaggatt?aggaggckcc?aacattcaac?????60

gtccacctca?gcagcagttg?aatcttttga?agggagaact?acttactccc?tattgccatg????120

gtttttccac?tttcatctgg?ggttgcaggg?gaagggtggg?ggtggggtgg?gaggagaagg????180

gacataacct?tgaaaaggac?tgtattaatc?accttctttg?taatcccttc?acagtcccag????240

gtttagtgga?aaactgctgt?aaacacagga?gacacagttt?aacaatgcaa?cttttaatga????300

ctgttttcat?tttccttaac?ctactaatag?tttgtggatc?tgatgagcag?gagtgggtgg????360

gtgagaaaaa?ctctgaatgg?gtttagccaa?tcactgtact?gcatccaaac?cagaaacgtg????420

tcacctgcgt?gacagtgggc?attcctctag?gcaaggtgca?gtaggaaatg?ctgcccacct????480

cagacgtcac?ccagccccct?ctcagtggtg?aagcttctgt?ttagaacacc?aaagatagga????540

ctagatacta?cttactttct?catataacct?ggtagacact?tacttgatga?tgtttttatt????600

tttaccttta?tttctaagtg?agaggaaatg?ctgatgtatc?ytttcatcca?actaaccaga????660

aaaggtgatg?ttctcttttc?aaaaagggaa?gtaagcaaac?tcagattgcc?aactcctata????720

tttatggatg?ctatacattg?cttatttaat?acacagttaa?cagtaatggt?gagttttaat????780

tctctccgta?gcgcctttgg?taattcacaa?tagtgatgga?tctaatggtt?ctagcatttt?????840

aagaaaccta?gacaagtaaa?attattctct?ttatgatttc?atgaaaaggt?acaacagaat?????900

aacccatgat?gaacttacct?ggattatgag?acgggagaag?caaaatctaa?atctattttg?????960

ctatagttat?actacaattt?aaagaacaac?aacaaagcag?gctctcttgt?ctctttcttt????1020

ytctctctgt?ctctctctcc?attgtgtatg?agtttctgtg?aaagatctaa?ataccatgtt????1080

cctcaaatga?agcttatgtg?ttactccagg?taatacgttt?tgacatagga?tggttggctg????1140

aagtgctttt?ctttgacatc?agcgycgc???????????????????????????????????????1168

<210>21

<211>1838

<212>DNA

<213>porcine

<220>

<221>misc_feature

<222>(725)..(725)

<223>n?can?be?any?nucleotide

<400>21

aaaaagtttc?tgggctagca?cctgttcatg?ggcctccttg?agtggccctg?ggttgggctc?????60

tgcctccact?ctctaaaagg?aaattgaagc?ccaagaagtt?gacagtgttg?aggagttggt????120

gcagagtgac?tcagagccct?gattctgtcc?cacccctccc?cccaaaggtc?acgtgaggtt????180

aaaaggccac?cctggcactt?tgtgcgcccc?agggagcttg?gcccgtcagg?ctgtggggac????240

cacctgttat?atggtggaga?tcttggtgtc?tgttacaggg?gggcagctgt?ccccaagtga????300

ggggcagcgg?ctggtggtga?agcccagtta?cttccttttc?aggggggaga?ggaaaggaat????360

tgaggtcgat?ccctggcctt?tagatggcag?gcagtttgtg?tacctgggcc?tccggcttcc????420

ccgtctgtag?gtggagagac?ctggcggagc?caggggtcat?gagaagtcca?atgggtgctg????480

gactcgagct?gcctcatgga?gggccctcag?ctcgtgggga?acttgtcctc?ttcatctggt????540

cctttggcct?ctcccagcck?cctgttagcg?gcggtcatgg?ttgcgggggg?atcagaaggg????600

gtgttgggtt?actggaccac?gcgcagcctg?gggaaaccat?agctgacgtg?cctttgctgc????660

ccagagcctg?tgctgcatgt?agcagctttt?tattctgtct?tgggttagta?caatttcagt?????720

ggcantaatg?ggcagggatc?tggggctcca?agatctggac?agaatcctct?gggggaggca?????780

gcctggaggt?cccttctgtt?tggggggatg?tcctctccca?cctcctgcat?cgccctggac?????840

actggcacgt?ccttcattgt?acattgttca?gtttttttta?ctgtcacagg?gaagcaaggg?????900

gagaggcctt?gcaaaggatg?ttcagactgg?gaacctgaat?ccccagggct?gtgcctgcca?????960

tgattcctgt?ggattctgga?gtggggctgt?cggggtgggg?gtggggtggg?gcagagactg????1020

tctggtgaaa?gaggtgggac?actggtgtct?atgccctgac?cgttccatct?gtctttgcag????1080

aaggagccca?gcgaagtgcc?aacacctaaa?agacctcggg?gccgaccaaa?ggggagcaaa????1140

aacaagggcg?ccgccaagac?ccgggtgagg?cttgaagggg?tggctcctgg?tggagggaag????1200

tgggaagtaa?cccccagccc?cctgcaagca?gctgagggag?gtctgggaag?gggtgggctt????1260

gtcctgattc?tctgcatgcc?ctttctctgg?tacgtgggcc?cgatgggtct?tggctagttg????1320

aggaaagtgg?ggtgatggcc?gaggcctaac?ttctagggcc?ttgtcttgcc?caggacactg????1380

gggaagtcaa?gtcagatgtc?ccagagcttt?cctggtctgg?agggaggcca?gttgggcaga????1440

atggagggct?gttccccctg?ggctgagatg?tcacctcccc?cccaacccca?ggccgcctgg????1500

gtcctgaggg?tgggggagca?ggcaaggcca?gatctacagt?ggcattggcc?tttggagaag????1560

ttgttttgtt?ttttatttta?ttttttctaa?gacacgactc?atatcctctg?agtcacgggt????1620

gaaggaggga?gtgggggcgt?gtgtgtgtat?gttggggtgg?ggggcggtgt?ggcyggccag????1680

tcatccccag?ctggactccg?gtgggcctgc?tgggctgaga?gtcccggctg?cccctccctg????1740

ctygccctcg?ccctccaggg?cactggtcac?tgcggggcac?ccgccattgg?gtgagcactg????1800

tcagggacat?ttttgacaat?ggatcttttc?tctgcaca????????????????????????????1838

<210>22

<211>2484

<212>DNA

<213>porcine

<400>22

ctggtaggag?tcaggtgggt?gtcaaacctt?tgcctctgtt?tacttctttg?ggctagggag?????60

gtgccaggcg?tcttggcagc?tgagagcaga?actggcgggg?cgcagagtcc?ttcctgtaca????120

tgtgtgttct?gtccacacgt?gcacatacct?ctcagaggag?tgcgagcctc?tgccagggtc????180

caagccgaag?ggaccctgtg?gtcaggcagg?ccagtgctca?caccggggta?aagcacccag????240

cctttgtttc?cacctcattg?gggcacagtg?catttgtgaa?acgtggtaaa?ggtgaattat?????300

agaaacgcaa?aatgacacat?gtgtaagccc?ggggttttca?cttgttatat?tcctcacaca?????360

cagtattagc?ctgttagttt?actccaaagg?tttctaatta?cttaacttac?tgtccatttc?????420

tctagttcct?cattccagta?cagtaaaaag?tttctgggct?agcacctgtt?catgggcctc?????480

cttgagtggc?cctgggttgg?gctctgcctc?cactctctaa?aaggaaattg?aagcccaaga?????540

agttgacagt?gttgaggagt?tggtgcagag?tgactcagag?ccctgattct?gtcccacccc?????600

tccccccaaa?ggtcacgtga?ggttaaaagg?ccaccctggc?actttgtgcg?ccccagggag?????660

cttggcccgt?caggctgtgg?ggaccacctg?ttatatggtg?gagatcttgg?tgtctgttac?????720

aggggggcag?ctgtccccaa?gtgaggggca?gcggctggtg?gtgaagccca?gttacttcct?????780

tttcaggggg?gagaggaaag?gaattgaggt?cgatccctgg?cctttagatg?gcaggcagtt?????840

tgtgtacctg?ggcctccggc?ttccccgtct?gtaggtggag?agacctggcg?gagccagggg?????900

tcatgagaag?tctaatgggt?gctggactcg?agctgcctca?tggagggccc?tcagctcgtg?????960

gggaacttgt?cctcttcatc?tggtcctttg?gcctctccca?gcctcctgtt?agcggcggtc????1020

atggttgtgg?ggggatcaga?aggggtgttg?ggttactgga?ccacacgcag?cctggggaaa????1080

ccatagctga?cgtgcctttg?ctgcccagag?cctgtgctgc?atgtagcagc?tttttattct????1140

gtcttgggct?tagtacaatt?tcagtgacac?taatgggcag?ggatctgggg?ctccaagatc????1200

tggacagaat?cctctggggg?aggcagcctg?gaggtccctt?ctgtttgggg?ggatgtcctc????1260

tcccacctcc?tgcatcgccc?tggacactgg?cacgtccttc?attgtacatt?gttcagtttt????1320

cgtgactcta?caaggtaggt?cttgtcacct?ttggcagatg?aggggaactg?tagctcaagc????1380

ataaagcgtc?ttgcccaaga?tcttagacct?aggcaatggc?agagctggga?tgcgaacaga????1440

gcagcctaag?aaggggtttc?tgtccccatg?acacccttcc?caatgggctt?ctagcctctt????1500

gtcattttac?tgtcacaggg?aagcaagggg?agaggccttg?caaaggatgt?tcagactggg????1560

aacctgaatc?cccagggctg?tgcctgccat?gattcctgtg?gattctggag?tggggctgtc????1620

ggggtggggg?tggggtgggg?cagagactgt?ctggtgaaag?aggtgggaca?ctggtgtcta????1680

tgccctgacc?gttccatctg?tctttgcaga?aggagcccag?cgaagtgcca?acacctaaaa????1740

gacctcgggg?ccgaccaaag?gggagcaaaa?acaagggcgc?tgccaagacc?cgggtgaggc????1800

ttgaaggggt?ggctcctggt?ggagggaagt?gggaagtaac?cccccgcccc?ctgcaagcag????1860

ctgagggagg?tctgggaagg?ggtgggcttg?tcctgattct?ctgcatgccc?tttctctggt????1920

acgtgggccc?gatgggtctt?ggctagttga?ggaaagtggg?gtgatggccg?aggcctaact????1980

tctagggcct?tgtcttgccc?aggacactgg?ggaagtcaag?tcagatgtcc?cagagctttc????2040

ctggtctgga?gggaggccag?ttgggcagaa?tggagggctg?ttccccctgg?gctgagatgt????2100

cacctccccc?ccaaccccag?gccgcctggg?tcctgagggt?gggggagcag?gcaaggccag????2160

atctacagtg?gcattggcct?ttggagaagt?tgttttgttt?tttattttat?tttttctaag????2220

acacgactca?tatcctctga?gtcacgggtg?aaggagggag?tgggggcgtg?tgtgtgtatg????2280

ttggggtggg?gggcggtgtg?gccggccagt?catccccagc?tggactccgg?tgggcctgct????2340

gggctgagag?tcccggctgc?ccctccctgc?tygccctcgc?cctccagggc?actggtcact????2400

gcggggcacc?cgccattggg?tgagcactgt?cagggacatt?tttgacaatg?gatcttttct????2460

ctgaccatct?agaaaaccac?caca???????????????????????????????????????????2484

<210>23

<211>2484

<212>DNA

<213>porcine

<400>23

ctggtaggag?tcaggtgggt?gtcaaacctt?tgcctctgtt?tacttctttg?ggctagggag?????60

gtgccaggcg?tcttggcagc?tgagagcaga?actggcgggg?cgcagagtcc?ttcctgtaca????120

tgtgtgttct?gtccacacgt?gcacatacct?ctcagaggag?tgcgagcctc?tgccagggtc????180

caagccgaag?ggacccggtg?gtcaggcagg?ccagtgctca?caccggggta?aagcacccag????240

cctttgtttc?cacctcatcg?gggcacagtg?catttgtgaa?acgtggtaaa?ggtgaattat????300

agaaacgcga?aatgacacat?gtgtaagccc?ggggttttca?cttgttatat?tcctcacaca????360

cagtattagc?ctgttagttt?actccaaagg?tttctaatta?cttaacttac?tgtccatttc????420

tctagttcct?cattccagta?cagtaaaaag?tttctgggct?agcacctgtt?catgggcctc????480

cttgagtggc?cctgggttgg?gctctgcctc?cactctctaa?aaggaaattg?aagcccaaga????540

agttgacagt?gttgaggagt?tggtgcagag?tgactcagag?ccctgattct?gtcccacccc????600

tccccccaaa?ggtcacgtga?ggttaaaagg?ccaccctggc?actttgtgcg?ccccagggag????660

cttggcccgt?caggctgtgg?ggaccacctg?ttatatggtg?gagatcttgg?tgtctgttac????720

aggggggcag?ctgtccccaa?gtgaggggca?gcggctggtg?gtgaagccca?gttacttcct????780

tttcaggggg?gagaggaaag?gaattgaggt?cgatccctgg?cctttagatg?gcaggcagtt????840

tgtgtacctg?ggcctccggc?ttccccgtct?gtaggtggag?agacctggcg?gagccagggg????900

tcatgagaag?tctaatgggt?gctggactcg?agctgcctca?tggagggccc?tcagctcgtg?????960

gggaacttgt?cctcttcatc?tggtcctttg?gcctctccca?gcctcctgtt?agcggcggtc????1020

atggttgtgg?ggggatcaga?aggggtgttg?ggttactgga?ccacgcgcag?cctggggaaa????1080

ccatagctga?cgtgcctttg?ctgcccagag?cctgtgctgc?atgtagcagc?tttttattct????1140

gtcttgggct?tagtacaatt?tcagtggcac?taatgggcag?ggatctgggg?ctccaagatc????1200

tggacagaat?cctctggggg?aggcagcctg?gaggtccctt?ctgtttgggg?ggatgtcctc????1260

tcccacctcc?tgcatcgccc?tggacactgg?cacgtccttc?attgtacatt?gttcagtttt????1320

cgtgactctg?caaggtaggt?cttgtcacct?ttggcagatg?aggggaactg?tagctcaagc????1380

ataaagcatc?ttgcccaaga?tcttagacct?aggcaatggc?agagctggga?tgcgaacaga????1440

gcagcctaag?aaggggtttc?tgtccccatg?acacccttcc?caatgggctt?ctagcctctt????1500

gtcattttac?tgtcacaggg?aagcaagggg?agaggccttg?caaaggatgt?tcagactggg????1560

aacctgaatc?cccagggctg?tgcctgccat?gattcctgtg?gattctggag?tggggctgtc????1620

ggggtggggg?tggggtgggg?cagagactgt?ctggtgaaag?aggtgggaca?ctggtgtcta????1680

tgccctgacc?gttccatctg?tctttgcaga?aggagcccag?cgaagtgcca?acacctaaaa????1740

gacctcgggg?ccgaccaaag?gggagcaaaa?acaagggcgc?cgccaagacc?cgggtgaggc????1800

ttgaaggggt?ggctcctggt?ggagggaagt?gggaagtaac?cccccgcccc?ctgcaagcag????1860

ctgagggagg?tctgggaagg?ggtgggcttg?tcctgattct?ctgcatgccc?tttctctggt????1920

acgtgggccc?gatgggtctt?ggctagttga?ggaaagtggg?gtgatggccg?aggcctaact????1980

tctagggcct?tgtcttgccc?aggacactgg?ggaagtcaag?tcagatgtcc?cagagctttc????2040

ctggtctgga?gggaggccag?ttgggcagaa?tggagggctg?ttccccctgg?gctgagatgt????2100

cacctccccc?ccaaccccag?gccgcctggg?tcctgagggt?gggggagcag?gcaaggccag????2160

atctacagtg?gcattggcct?ttggagaagt?tgttttgttt?tttattttat?tttttctaag????2220

acacgactca?tatcctctga?gtcacgggtg?aaggagggag?tgggggcgtg?tgtgtgtatg????2280

ttggggtggg?gggcggtgtg?gctggccagt?catccccagc?tggactccgg?tgggcctgct????2340

gggctgagag?tcccggctgc?ccctccctgc?tcgccctcgc?cctccagggc?actggtcact????2400

gcggggcacc?cgccattggg?tgagcactgt?cagggacatt?tttgacaatg?gatcttttct????2460

ctgaccatct?agaaaaccac?caca???????????????????????????????????????????2484

<210>24

<211>1950

<212>DNA

<213>porcine

<400>24

ctggtaggag?tcaggtgggt?gtcaaacctt?tgcctctgtt?tacttctttg?ggctagggag??????60

gtgccaggcg?tcttggcagc?tgagagcaga?actggcgggg?cgcagagtcc?ttcctgtaca?????120

tgtgtgttct?gtccacacgt?gcacatacct?ctcagaggag?tgcgagcctc?tgccagggtc?????180

caagccgaag?ggacccggtg?gtcaggcagg?ccagtgctca?caccggggta?aagcacccag?????240

cctttgtttc?cacctcatcg?gggcacagtg?catttgtgaa?acgtggtaaa?ggtgaattat?????300

agaaacgcga?aatgacacat?gtgtaagccc?ggggttttca?cttgttatat?tcctcacaca?????360

cagtattagc?ctgttagttt?actccaaagg?tttctaatta?cttaacttac?tgtccatttc?????420

tctagttcct?cattccagta?cagtaaaaag?tttctgggct?agcacctgtt?catgggcctc?????480

cttgagtggc?cctgggttgg?gctctgcctc?cactctctaa?aaggaaattg?aagcccaaga?????540

agttgacagt?gttgaggagt?tggtgcagag?tgactcagag?ccctgattct?gtcccacccc?????600

tccccccaaa?ggtcacgtga?ggttaaaagg?ccaccctggc?actttgtgcg?ccccagggag?????660

cttggcccgt?caggctgtgg?actttgtgcg?ccccagggag?cttggcccgt?caggctgtgg?????720

ggaccacctg?ttatatggtg?gagatcttgg?tgtctgttac?aggggggcag?ctgtccccaa?????780

gtgaggggca?gcggctggtg?gtgaagccca?gttacttcct?tttcaggggg?gagaggaaag?????840

gaattgaggt?cgatccctgg?cctttagatg?gcaggcagtt?tgtgtacctg?ggcctccggc?????900

ttccccgtct?gtaggtggag?agacctggcg?gagccagggg?tcatgagaag?tctaatgggt?????960

gctggactcg?agctgcctca?tggagggccc?tcagctcgtg?gggaacttgt?cctcttcatc????1020

tggtcctttg?gcctctccca?gcctcctgtt?agcggcggtc?atggttgtgg?ggggatcaga????1080

aggggtgttg?ggttactgga?ccacgcgcag?cctggggaaa?ccatagctga?cgtgcctttg????1140

ctgcccagag?cctgtgctgc?atgtagcagc?tttttattct?gtcttgggct?tagtacaatt????1200

tcagtggcac?taatgggcag?ggatctgggg?ctccaagatc?tggacagaat?cctctggggg????1260

aggcagcctg?gaggtccctt?ctgtttgggg?ggatgtcctc?tcccacctcc?tgcatcgccc????1320

tggacactgg?cacgtccttc?attgtacatt?gttcagtttt?cgtgactctg?caaggtaggt????1380

cttgtcacct?ttggcagatg?aggggaactg?tagctcaagc?ataaagcatc?ttgcccaaga????1440

tcttagacct?aggcaatggc?agagctggga?tgcgaacaga?gcagcctaag?aaggggtttc????1500

tgtccccatg?acacccttcc?caatgggctt?ctagcctctt?gtcattttac?tgtcacaggg????1560

aagcaagggg?agaggccttg?caaaggatgt?tcagactggg?cacctgaatc?cccagggctg????1620

tgcctgccat?gattcctgtg?gattctggag?tggggctgtc?ggggtggggg?tggggtgggg????1680

cagagactgt?ctggtgaaag?aggtgggaca?ctggtgtcta?tgccctgacc?gttccatctg????1740

tctttgcaga?aggagcccag?cgaagtgcca?acacctaaaa?gacctcgggg?ccgaccaaag????1800

ttgaaggggt?ggctcctggt?ggagggaagt?gggaagtaac?cccccgcccc?ctgcaagcag????1860

ctgagggagg?tctgggaagg?ggtgggcttg?tcctgattct?ctgcatgccc?tttctctggt????1920

acgtgggccc?gatgggtctt?ggctagttga?????????????????????????????????????1950

<210>25

<211>2404

<212>DNA

<213>porcine

<400>25

ctggtaggag?tcaggtgggt?gtcaaacctt?tgcctctgtt?tacttctttg?ggctagggag?????60

gtgccaggcg?tcttggcagc?tgagagcaga?actggcgggg?cgcagagtcc?ttcctgtaca????120

tgtgtgttct?gtccacacgt?gcacatacct?ctcagaggag?tgcgagcctc?tgccagggtc????180

caagccgaag?ggacccggtg?gtcaggcagg?ccagtgctca?caccggggta?aagcacccag????240

cctttgtttc?cacctcatcg?gggcacagtg?catttgtgaa?acgtggtaaa?ggtgaattat????300

agaaacgcga?aatgacacat?gtgtaagccc?ggggttttca?cttgttatat?tcctcacaca????360

cagtattagc?ctgttagttt?actccaaagg?tttctaatta?cttaacttac?tgtccatttc????420

tctagttcct?cattccagta?cagtaaaaag?tttctgggct?agcacctgtt?catgggcctc????480

cttgagtggc?cctgggttgg?gctctgcctc?cactctctaa?aaggaaattg?aagcccaaga????540

agttgacagt?gttgaggagt?tggtgcagag?tgactcagag?ccctgattct?gtcccacccc????600

tccccccaaa?ggtcacgtga?ggttaaaagg?ccaccctggc?actttgtgcg?ccccagggag????660

cttggcccgt?caggctgtgg?ggaccacctg?ttatatggtg?gagatcttgg?tgtctgttac????720

aggggggcag?ctgtccccaa?gtgaggggca?gcggctggtg?gtgaagccca?gttacttcct????780

tttcaggggg?gagaggaaag?gaattgaggt?cgatccctgg?cctttagatg?gcaggcagtt????840

tgtgtacctg?ggcctccggc?ttccccgtct?gtaggtggag?gctggactcg?agctgcctca????900

tggagggccc?tcagctcgtg?gggaacttgt?cctcttcatc?tggtcctttg?gcctctccca????960

gcctcctgtt?agcggcggtc?atggttgtgg?ggggatcaga?aggggtgttg?ggttactgga????1020

ccacgcgcag?cctggggaaa?ccatagctga?cgtgcctttg?ctgcccagag?cctgtgctgc????1080

atgtagcagc?tttttattct?gtcttgggct?tagtacaatt?tcagtggcac?taatgggcag????1140

ggatctgggg?ctccaagatc?tggacagaat?cctctggggg?aggcagcctg?gaggtccctt????1200

ctgtttgggg?ggatgtcctc?tcccacctcc?tgcatcgccc?tggacactgg?cacgtccttc????1260

attgtacatt?gttcagtttt?cgtgactctg?caaggtaggt?cttgtcacct?ttggcagatg????1320

aggggaactg?tagctcaagc?ataaagcatc?ttgcccaaga?gcagcctaag?aaggggtttc????1380

tgtccccatg?acacccttcc?caatgggctt?ctagcctctt?gtcattttac?tgtcacaggg????1440

aagcaagggg?agaggccttg?caaaggatgt?tcagactggg?aacctgaatc?cccagggctg????1500

tgcctgccat?gattcctgtg?gattctggag?tggggctgtc?ggggtggggg?tggggtgggg????1560

cagagactgt?ctggtgaaag?aggtgggaca?ctggtgtcta?tgccctgacc?gttccatctg????1620

tctttgcaga?aggagcccag?cgaagtgcca?acacctaaaa?gacctcgggg?ccgaccaaag????1680

gggagcaaaa?acaagggcgc?cgccaagacc?cgggtgaggc?ttgaaggggt?ggctcctggt????1740

ggagggaagt?gggaagtaac?cccccgcccc?ctgcaagcag?ctgagggagg?tctgggaagg????1800

ggtgggcttg?tcctgattct?ctgcatgccc?tttctctggt?acgtgggccc?gatgggtctt????1860

ggctagttga?ggaaagtggg?gtgatggccg?aggcctaact?tctagggcct?tgtcttgccc????1920

aggacactgg?ggaagtcaag?tcagatgtcc?cagagctttc?ctggtctgga?gggaggccag????1980

ttgggcagaa?tggagggctg?ttccccctgg?gctgagatgt?cacctccccc?ccaaccccag????2040

gccgcctggg?tcctgagggt?gggggagcag?gcaaggccag?atctacagtg?gcattggcct????2100

ttggagaagt?tgttttgttt?tttattttat?tttttctaag?acacgactca?tatcctctga????2160

gtcacgggtg?aaggagggag?tgggggcgtg?tgtgtgtatg?ttggggtggg?gggcggtgtg????2220

gcyggccagt?catccccagc?tggactccgg?tgggcctgct?gggctgagag?tcccggctgc????2280

ccctccctgc?tcgccctcgc?cctccagggc?actggtcact?gcggggcacc?cgccattggg????2340

tgagcactgt?cagggacatt?tttgacaatg?gatcttttct?ctgaccatct?agaaaaccac????2400

caca?????????????????????????????????????????????????????????????????2404

<210>26

<211>1103

<212>DNA

<213>porcine

<400>26

tggtaggtga?agaggctggg?aaagcagcgg?cggcagcttg?gctcctctgg?ggaggctctt??????60

aagactgggt?ggagccccct?gtcttgtgtg?tccgatacag?agagacagac?ttttctgacc?????120

tgcggggcca?cgagcgctgg?gacttcgcac?ttagccaggc?ccaggaggga?ggatctgccc?????180

aggcccgggc?tgttgggggg?ctgtgtccac?gagcagtgaa?gcggcccagg?tgcaaagcag?????240

cccgcactcc?ttgccctgcc?ccactgggcc?tggtctcacg?ctctcttccc?tcgacttcta?????300

gaccaggctc?agaacttcta?gggcgaacga?aatgagggag?ggatcaggtc?cattttgacc?????360

ctggggcagg?aggaagcagc?agcagtggag?ccccatcctg?gggcgggtgg?tgtccgtgtt?????420

gggtggcagc?tgaggtggga?cacctcctca?ccaggaggca?ccatccactt?ctgggcccca?????480

aatagctga?gccagaggct?gcctgcaggg?ggcgcactgc?agtgagggcc?accagggcct?????540

tcctctaggc?ctcccctgct?ccccacctgt?gctccctggg?gccttctttc?ctccaccgcc?????600

accaccatca?ctgggtgcag?ggggtgaggg?ggtgagggga?tgctggcagg?gccccaagag?????660

tgagtaacag?gaaacaagtt?gttttggagt?ttgtgcctgg?cacgggggcc?cccatgtggt?????720

gtcccaacat?tccggcccag?tgagtgagcc?ccacacttcc?ccttcctccc?cgccctggcc?????780

tggggtcagc?ccgcggccag?cctgttgtgg?ccgctccaca?gcccagcagc?tgcccctgcg?????840

ggccaaggcc?acctgggtcc?ccggcaccca?ccagtgcagt?gagggggctc?attgcaccct?????900

ccaggcaccc?tgtcccattt?cctcccctga?ccacctccct?gctcccccca?ccacccagcc?????960

agtcacttcc?tccagcctag?tgctgccctg?ggccctgcca?ggtccctgcc?ttgggcctgg????1020

gggccaaggg?cctggctcgg?tgagagcagc?ccatgtgtgt?ggtttttttc?cctcccttta????1080

aattcttcct?tttttatgaa?tga????????????????????????????????????????????1103

<210>27

<211>1103

<212>DNA

<213>porcine

<400>27

tggtaggtga?agaggctggg?aaagcagcgg?cggcagcttg?gctcctctgg?ggaggctctt??????60

aagactgggt?ggagccccct?gtcttgtgtg?tccgatacag?agagacagac?ttttctgacc?????120

tgcggggcca?cgagcgctgg?gacttcgcac?ttagccaggc?ccaggaggga?ggatctgccc?????180

aggcccgggc?tgttgggggg?ctgtgtccac?gagcagtgaa?gcggcccagg?tgcaaagcag?????240

cccgcactcc?ttgccctgcc?ccactgggcc?tggtctcacg?ctctcttccc?tcgacttcta?????300

gaccaggctc?agaacttcta?gggcgaacga?aatgagggag?ggatcaggtc?cattttgacc?????360

ctggggcagg?aggaagcagc?agcagtggag?ccccatcctg?gggcgggtgg?tgtccgtgtt?????420

gggtggcagc?tgaggtggga?cacctcctca?ccaggaggca?ccatccactt?ctgggcccca?????480

aatagctgag?gccagaggct?gcctgcaggg?ggcgcactgc?agtgagggcc?accagggcct?????540

tcctctaggc?ctcccctgct?ccccacctgt?gctccctggg?gccttctttc?ctccaccgcc?????600

accaccatca?ctgggtgcag?ggggtgaggg?ggtgagggga?tgctggcagg?gccccaagag?????660

tgagtaacag?gaaacaagtt?gttttggagt?ttgtgcctgg?cacgggggcc?cccatgtggt?????720

gtcccaacat?tccggcccag?tgagtgagcc?ccacacttcc?ccttcctccc?cgccctggcc?????780

tggggtcagc?ccgcggccag?cctgttgtgg?ccgctccaca?gcccagcagc?tgcccctgcg?????840

ggccaaggcc?acctgggtcc?ccggcaccca?ccagtgcagt?gagggggctc?attgcaccct?????900

ccaggcaccc?tgtcccattt?cctcccctga?ccacctccct?gctcccccca?ccacccagcc?????960

agtcacttcc?tccagcctag?tgctgccctg?ggccctgcca?ggtccctgcc?ttgggcctgg????1020

gggccaaggg?cctggctcgg?tgagagcagc?ccatgtgtgt?ggtttttttc?cctcccttta????1080

aattcttcct?tttttatgaa?tga????????????????????????????????????????????1103

<210>28

<211>1102

<212>DNA

<213>porcine

<220>

<221>misc_feature

<222>(949)..(949)

<223>n?is?any?nucleotide

<220>

<221>misc_feature

<222>(957)..(957)

<223>n?is?any?nucleotide

<400>28

tggtaggtga?agaggctggg?aaagcagcgg?cggcagcttg?gctcctctgg?ggaggctctt??????60

aagactgggt?ggagccccct?gtcttgtgtg?tccgatacag?agagacagac?ttttctgacc?????120

tgmggggcca?cgagcgctgg?gacttcgcac?ttagccaggc?ccaggaggga?ggatctgccc?????180

aggcccgggc?tgttgggggg?ctgtgtccac?gagcagtgaa?gcggcccagg?tgcaaagcag?????240

cccgcactcc?ttgccctgcc?ccactgggcc?tggtctcacg?ctctcttccc?tcgacttcta?????300

gaccaggctc?agaacttcta?gggcgaacga?aatgagggag?ggatcaggtc?cattttgacc?????360

ctggggcagg?aggaagcagc?agcagtggag?ccccatcctg?gggcgggtgg?tgtccgtgtt?????420

gggtggcagc?tgaggtggga?cacctcctca?ccaggaggca?ccatccactt?ctgggcccca?????480

aatagctgag?gccagaggct?gcctgcaggg?ggcgcactgc?agtgagggcc?accagggcct?????540

tcctctaggc?ctcccctgct?ccccacctgt?gctccctggg?gccttctttc?ctccaccgcc?????600

accaccatca?ctgggtgcag?ggggtgaggg?ggtgagggga?tgctggcagg?gccccaagag?????660

tgagtaacag?gaaacaagtt?gttttggagt?ttgtgcctgg?cacgggggcc?cccatgtggt?????720

gtcccaacat?tccggcccag?tgagtgagcc?ccacacttcc?ccttcctccc?cgccctggcc?????780

tggggtagcc?cgcggccagc?ctgttgtggc?cgctccacag?cccagcagct?gcccctgcgg?????840

gccaaggcca?cctgggtccc?cggcacccac?cagtgcagtg?agggggctca?ttgcaccctc?????900

caggcaccct?gtcccatttc?ctcccctgac?cacctccctg?ctccccccnc?cacccancca?????960

gtcacttcct?ccagcctagt?gctgccctgg?gccctgccag?gtccctgcct?tgggcctggg????1020

ggccaagggc?ctggctcggt?gagagcagcc?catgtgtgtg?gtttttttcc?ctccctttaa????1080

attcttcctt?ttttatgaat?ga?????????????????????????????????????????????1102

<210>29

<211>1059

<212>DNA

<213>porcine

<400>29

tggtaggtga?agaggctggg?aaagcagcgg?cggcagcttg?gctcctctgg?ggaggctctt??????60

aagactgggt?ggagccccct?gtcttgtgtg?tccgatacag?agagacagac?ttttctgacc?????120

tgcggggcca?cgagcgctgg?gacttcgcac?ttagccaggc?ccaggaggga?ggatctgccc?????180

aggcccgggc?tgttgggggg?ctgtgtccac?gagcagtgaa?gcggcccagg?tgcaaagcag?????240

cccgcactcc?ttgccctgcc?ccactgggcc?tggtctcacg?ctctcttccc?tcgacttcta?????300

gaccaggctc?agaacttcta?gggcgaacga?aatgagggag?ggatcaggtc?cattttgacc?????360

ctggggcagg?aggaagcagc?agcagtggag?ccccatcctg?gggcgggtgg?tgtccgtgtt?????420

gggtggcagc?tgaggtggga?cacctcctca?ccaggaggca?ccatccactt?ctgggcccca?????480

aatagctgag?gccagaggct?gcctgcaggg?ggcgcactgc?agtgagggcc?accagggcct?????540

tcctctaggc?ctcccctgct?ccccacctgt?gctccctggg?gccttctttc?ctccaccgcc?????600

accaccatca?ctgggtgcag?ggggtgaggg?ggtgagggga?tgctggcagg?gccccaagag?????660

tgagtaacag?gaaacaagtt?gttttggagt?ttgtgctggc?acgggggccc?ccatgtggtg?????720

tcccaacatt?ccggcccagt?gagtgagccc?cacacttccc?tttcctcccc?gccctggcct?????780

ggggtcagcc?cgcggccagg?gccaaggcca?cctgggtccc?cggcacccac?cagtgcagtg?????840

agggggctca?ttgcaccctc?caggcaccct?gtcccatttc?ctcccctgac?cacctccctg?????900

ctccccccac?cacccagcca?gtcattcctc?cagctagtgc?tgccctgggc?cctgccaggt?????960

ccctgccttg?ggcctggggg?ccaagggctg?gctcggtgag?agcagcccat?gtgtgtggtt????1020

tttttccctc?cctttaaatt?cttccttttt?tatgaatga???????????????????????????1059

<210>30

<211>1103

<212>DNA

<213>porcine

<400>30

tggtaggtga?agaggctggg?aaagcagcgg?cggcagcttg?gctcctctgg?ggaggctctt?????60

aagactgggt?ggagccccct?gtcttgtgtg?tccgatacag?agagacagac?ttttctgacc????120

tgcggggcca?cgagcgctgg?gacttcgcac?ttagccaggc?ccaggaggga?ggatctgccc????180

aggcccgggc?tgttgggggg?ctgtgtccac?gagcagtgaa?gcggcccagg?tgcaaagcag????240

cccgcactcc?ttgccctgcc?ccactgggcc?tggtctcacg?ctctcttccc?tcgacttcta????300

gaccaggctc?agaacttcta?gggcgaacga?aatgagggag?ggatcaggtc?cattttgacc????360

ctggggcagg?aggaagcagc?agcagtggag?ccccatcctg?gggcgggtgg?tgtccgtgtt????420

gggtggcagc?tgaggtggga?cacctcctca?ccaggaggca?ccatccactt?ctgggcccca????480

aatagctgag?gccagaggct?gcctgcaggg?ggcgcactgc?agtgagggcc?accagggcct?????540

tcctctaggc?ctcccctgct?ccccacctgt?gctccctggg?gccttctttc?ctccaccgcc?????600

accaccatca?ctgggtgcag?ggggtgaggg?ggtgagggga?tgctggcagg?gccccaagag?????660

tgagtaacag?gaaacaagtt?gttttggagt?ttgtgcctgg?cacgggggcc?cccatgtggt?????720

gtcccaacat?tccggcccag?tgagtgagcc?ccacacttcc?ccttcctccc?cgccctggcc?????780

tggggtcagc?ccgcggccag?cctgttgtgg?ccgctccaca?gcccagcagc?tgcccctgcg?????840

ggccaaggcc?acctgggtcc?ccggcaccca?ccagtgcagt?gagggggctc?attgcaccct?????900

ccaggcaccc?tgtcccattt?cctcccctga?ccacctccct?gctcccccca?ccacccagcc?????960

agtcacttcc?tccagcctag?tgctgccctg?ggccctgcca?ggtccctgcc?ttgggcctgg????1020

gggccaaggg?cctggctcgg?tgagagcagc?ccatgtgtgt?ggtttttttc?cctcccttta????1080

aattcttcct?tttttatgaa?tga????????????????????????????????????????????1103

Claims (31)

1, a kind of discriminating has the method for the genotypic animal that characterizes a kind of phenotypic character, described method comprises: obtain nucleic acid samples from described animal, whether have the genotype that is characterized as HMGA1 or HMGA2 gene pleiomorphism in the test sample or whether have the polymorphism chain with it, described genotype is the genotype that has shown with a kind of phenotypic character significant correlation; And described animal is associated with described phenotypic character according to the genotype that exists in the described animal.

2, the process of claim 1 wherein that contrasting definite described polymorphism through BLAST causes the amino acid of HMGA gene or its Equivalent to change.

3, the process of claim 1 wherein that described polymorphic position is in the HMGA1 gene.

4, the method for claim 3, wherein said genotype are NaeI or BanI polymorphism.

5, the process of claim 1 wherein that described polymorphism is positioned in the HMGA2 gene.

6, the method for claim 5, wherein said genotype are the HhaI polymorphisms.

7, the process of claim 1 wherein that described detection step is selected from in next group: restriction fragment length polymorphism (RFLP) analysis, micrometering preface, MALD-TOF, SINE, heteroduple analysis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel elec-trophoresis (TGGE) (TGGE).

8, the process of claim 1 wherein that described animal is a pig.

9, the method for claim 1, described method comprise that further amplification HMGA nucleotide sequence or its contain the method for the amount of described polymorphism part.

10, the method for claim 9, wherein said amplification may further comprise the steps: can the increase forward and the reverse primer in a zone of the HMGA nucleotide sequence that contains one or more polymorphism NaeI, BanI or HhaI site of selection.

11, the method for claim 9, wherein said forward and reverse primer are selected from next group: SEQ ID No:1 and 2,3 and 4,5 and 6,7 and 8,9 and 10,11 and 12,13 and 14,15 and 16, or 17 and 18.

12, a kind of method of screening animal with the animal of growth, fat quantity, meat and the feed conversion ratio proterties of determining more may to illustrate improvement comprises: obtain biological sample from described animal; Detect and whether exist in the described animal and the growth, fat quantity, meat of improvement, the genotype that the feed conversion ratio proterties is relevant, described genotypic being characterized as: a) polymorphism in the HMGA nucleotide sequence, described polymorphism cause one or more NaeI, BanI or HhaI site.

13, the method for claim 12, described method comprise that further amplification HMGA coding nucleotide sequence gene or its contain the step of the amount of described polymorphism part.

14, a kind of proteic nucleotide sequence of HMGA or allelotrope of encoding when expressing, described nucleotide sequence comprises by the variant due to NaeI, BanI or the HhaI pleomorphism site.

15, the HMGA albumen of claim 11.

16, a kind of discriminating has the method for the required genotypic animal of the phenotypic character that characterizes a kind of significant correlation, this method comprises: obtain the nucleic acid samples that comprises HMGA1 or HMGA2 gene from animal, this sample is used and BanI, the Restriction Enzyme that NaeI or HhaI recognition site are identical digests to obtain fragment, the fragment that digestion produces is separated from digest, whether there is BanI on the allelotrope of discriminating HMGA1 or HMGA2 gene, NaeI or HhaI site wherein exist described allelotrope to show that animal has the genotype of the phenotypic character that characterizes a kind of significant correlation.

17, a kind of selection has the method for the animal of required proterties, comprise the steps: from animal, to obtain nucleic acid samples, differentiate polymorphism, described polymorphism is to be characterized as the HMGA1 of BanI, NaeI or HhaI restriction site or the Nucleotide in the HMGA2 gene, selects to have the animal of the Nucleotide relevant with required proterties then.

18, the method for polymorphism in a kind of indirect selection HMGA gene, described method comprises: obtain nucleic acid samples from an animal, and be the polymorphism of BanI, NaeI or HhaI restriction site with known and dna marker diagnostic characteristics in HMGA1 or the HMGA2 gene HMGA gene-correlation, described dna marker further is known and the relevant mark of favourable proterties that is used for differentiating indirectly that Nucleotide replaces, and selects described animal according to the existence of Nucleotide replacement.

19, a kind of discriminating has the method for the required genotypic animal that characterizes phenotypic character, this method comprises: by from the animal of interested strain or kind, obtain sample, nucleic acid samples, the screening polymorphism of each animal of preparation determines that the HMGA gene genotype determines related between HMGA genotype and interested proterties from each sample, wherein the existence of this polymorphism shows that this animal has the genotype that characterizes favourable phenotypic character, calculates related between this HMGA genotype and the described proterties then.

20, a kind of method of selecting breeding with animal, described method comprises: obtain nucleic acid samples from described animal; Whether test sample HMGA1 or HMGA2 gene exist polymorphism, and described polymorphism has illustrated and the phenotypic character significant correlation; According to the Effect Estimation value of marker gene type, HMGA1 or HMGA2 genotype are used in the preference pattern then, thereby and according to the animal of the estimated value selection that draws as breeding.

21, a kind ofly isolate animal so that the method for homogeneity to be provided when butchering, this method comprises: obtain nucleic acid samples from described animal; Whether polymorphism relevant with meat in the test sample exists in the HMGA gene, according to the polymorphism that is present in the described animal described animal is isolated then.

22, a kind of screening more may produce the method for the animal of required growth, fat quantity, meat and feed conversion ratio, and this method comprises: obtain the genetic material sample from described animal; And detecting genotype existence relevant with the growth, fat quantity, meat and the feed conversion ratio that improve in described animal, described genotype has following feature: a) polymorphism in the HMGA gene.

23, the method for claim 22 wherein determines that with the BLAST contrast described polymorphism causes the amino acid of HMGA gene or its Equivalent to change.

24, the method for claim 22, wherein said polymorphic position is in HMGA1 or HMGA2 gene.

25, the method for claim 22, wherein said genotype are NaeI, BanI or HhaI polymorphism.

26, the method for claim 22, wherein said detection step is selected from next group: restriction fragment length polymorphism (RFLP) analysis, micrometering preface, MALD-TOF, SINE, heteroduple analysis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel elec-trophoresis (TGGE) (TGGE).

27, the method for claim 22, wherein said animal is a pig.

28, the method for claim 22 comprises that further amplification contains the HMGA nucleotide sequence of described polymorphism or the step of its part amount.

29, the method for claim 28, wherein said amplification may further comprise the steps: can the increase forward and the reverse primer in a zone of the HMGA nucleotide sequence that contains one or more NaeI, BanI or HhaI site of selection.

30, a coding proteic nucleotide sequence of HMGA or an allelotrope when expressing, described nucleotide sequence comprises SEQ ID NOS:19,20,21,22,23,24,25,26,27,28 or 29.

31, the HMGA albumen of claim 30.